Xylanse variants having altered sensitivity to xylanse inhibitors

ABSTRACT

The present invention relates to a variant xylanase polypeptide, or fragment thereof having xylanase activity, comprising one or more amino acid modifications such that the polypeptide or fragment thereof has an altered sensitivity to a xylanase inhibitor as compared with the parent enzyme.

FIELD OF THE INVENTION

[0001] The present invention relates to mutant xylanase enzymes havingan altered sensitivity to xylanase inhibitors. The present inventionalso relates to the use of these mutant enzymes in processing plantmaterials.

BACKGROUND TO THE INVENTION

[0002] For many years, endo-β-1,4-xylanases (EC 3.2.1.8) (referred toherein as xylanases) have been used for the modification of complexcarbohydrates derived from plant cell wall material. It is well known inthe art that the functionality of different xylanases (derived fromdifferent micro organisms or plants) differs enormously.

[0003] Comprehensive studies characterising the functionality ofxylanases have been done on well characterised and pure substrates(Kormelink et al., 1992). These studies show that different xylanaseshave different specific requirements with respect to substitution of thexylose backbone of the arabinoxylan (AX). Some xylanases require threeun-substituted xylose residues to hydrolyse the xylose backbone; othersrequire only one or two. The reasons for these differences inspecificity is thought to be due to the three dimensional structurewithin the catalytic domains, which in turn is dependent on the primarystructure of the xylanase, i.e. the amino acid sequence. However, thetranslation of these differences in the amino acid sequences intodifferences in the functionality of the xylanases, has up until now notbeen documented when the xylanase acts in a complex environment, such asplant material.

[0004] The xylanase substrates found in wheat (wheat flour), havetraditionally been divided into two fractions: The water un-extractableAX (WU-AX) and the water extractable AX (WE-AX). The WU-AX:WE-AX ratiois approx. 70:30 in wheat flour. There have been numerous explanationsas to why there are two different fractions of AX. The older literature(D'Appolonia and MacArthur (1976) and Montgomery and Smith (1955))describes quite high differences in the substitution degree betweenWE-AX and WU-AX. The highest degree of substitution was found in WE-AX.This was used to explain why some of the AX was extractable. The highdegree of substitution made the polymer soluble, compared to a lowersubstitution degree, which would cause hydrogen bonding between polymersand consequently precipitation.

[0005] The difference between the functionality of different xylanaseshas been thought to be due to differences in xylanase specificity andthereby their preference for the WU-AX or the WE-AX substrates.

[0006] In some applications (e.g. bakery) it is desirable to producehigh molecular weight (HMW) soluble polymers from the WU-AX fraction.Such polymers have been correlated to a volume increase in bread making(Rouau, 1993; Rouau et al., 1994 and Courtin et al., 1999).

[0007] In other applications it is desirable to modify the HMW WU-AX,making the molecular weight lower, reducing their hydrocolloid effectand hence water-binding in the product (crackers, flour separation,etc.)

[0008] These different applications require different functionalities ofthe xylanases used to do the job. As mentioned above, the difference infunctionality has been explained by the different substratespecificities of the xylanases.

SUMMARY OF THE INVENTION

[0009] By contrast to earlier studies, we have now shown that otherfactors are more important in determining xylanase functionality thanthe substrate specificity of the xylanases determined on purewell-characterised substrates. The data presented herein show thatendogenous xylanase inhibitors dictate the functionality of thexylanases currently used in, for example, wheat flour systems. Thismeans that a xylanase that normally modifies the WU-AX, giving increaseddough liquid viscosity in a wheat flour system, has a differentfunctionality if the endogenous xylanase inhibitor is absent in thewheat flour. Thus, our findings indicate that the design and applicationof uninhibited xylanases, for example, using site-directed mutagenesiscould be a way to mimic the absence of xylanase inhibitors in variousplant materials, giving new xylanases with completely new functionality.Such xylanases would be very effective in applications where a reductionin viscosity is required. The uninhibited xylanase would act rapidly onthe AX, and be primarily influenced by its specific activity, ratherthan by endogenous inhibitors. From our studies, we consider that theinhibitory effects are likely to be far more important than the specificactivity. Indeed our results show for the first time that there are 10to 50 fold differences in inhibition levels between the family 11xylanases.

[0010] Furthermore, we have gone on to design and test a series ofxylanases modified by site-directed mutagenesis to demonstrate thatxylanases can be produced that have reduced sensitivity to xylanaseinhibitors present in plant materials. In particular, we have identifieda number of residues in family 11 xylanases which influence the degreeof inhibition of the xylanase.

[0011] Thus, it will be possible to produce variant xylanases havingreduced sensitivity to xylanase inhibitors and hence alteredfunctionality. This will, for example, allow a reduction in the amountof xylanase required in a number of applications such as animal feed,starch production, bakery, flour separation (wetmilling) and, paper andpulp production.

[0012] Accordingly, the present invention provides a variant xylanasepolypeptide, or fragment thereof having xylanase activity, comprisingone or more amino acid modifications such that the polypeptide orfragment thereof has an altered sensitivity to a xylanase inhibitor ascompared with the parent enzyme.

[0013] Here, the “parent enzyme” is the xylanase enzyme from which thevariant xylanase enzyme is derived or derivable. With respect to theterm “derivable”, the variant need not necessarily be derived from theparent enzyme. Instead, the variant could be prepared, for example, byuse of recombinant DNA techniques that utilise nucleotide sequence(s)encoding said variant xylanase sequence—i.e. here the nucleotidesequence(s) are similar to mutated nucleotide sequence(s) but they arenot prepared by mutation of the parent nucleotide sequence(s). Thevariant may even be prepared by chemically modifying a parent enzyme.

[0014] For some embodiments the parent enzyme is the wild type enzyme.The term “wild type” is a term of art understood by skilled persons andincludes a phenotype that is characteristic of most of the members of aspecies occurring naturally and contrasting with the phenotype of amutant. Thus, in the present context, the wild type enzyme may be a formof the enzyme naturally found in most members of the relevant species.Generally, the relevant wild type enzyme in relation to the variantpolypeptides of the invention is the most closely related correspondingwild type enzyme in terms of sequence homology. For example, for theparticular mutant xylanases described in the examples, the correspondingwild type enzyme is the wild type B. subtilis xylanase A, morespecifically the wild type B. subtilis xylanase A published by Paice etal., 1986 and shown as SEQ I.D. 1. However, where a particular wild typesequence has been used as the basis for producing a variant polypeptideof the invention, this will be the corresponding wild type sequenceregardless of the existence of another wild type sequence that is moreclosely related in terms of amino acid sequence homology.

[0015] For some embodiments, preferably the variant polypeptide isderived from a family 11 xylanase.

[0016] One of our surprising findings is that in our studies so far amutation in the xylanase active site has no measurable effect oninhibition against the xylanase inhibitor. This is in direct contrast tothe mutation(s) that are made outside of the active site—which mutationsare discussed in more detail below.

[0017] In a preferred aspect the amino acid modification is of one ormore surface amino acid residues.

[0018] In a more preferred aspect the amino acid modification is of oneor more solvent accessible residues. Here, the solvent is water.

[0019] In a more preferred aspect the amino acid modification is of oneor more surface residues outside of the active site.

[0020] In a highly preferred aspect the amino acid modification is ofone or more surface residues outside of the active site and which is/areat least 8% solvent accessible. Here, the solvent is water.

[0021] In a highly preferred aspect the amino acid modification is ofone or more surface residues outside of the active site and which is/areat least 10% solvent accessible. Here, the solvent is water.

[0022] Solvent accessibility can be determined using Swiss-PdbViewer(version 3.5b1), which can be located via the internet athttp:///www.expasy.ch/spdbv/mainpage.html. The Swiss-PdbViewer ispresented by Glaxo Wellcome Experimental Research.

[0023] The surface amino acids of xylanase enzymes are determinable by aperson skilled in the art.

[0024] By way of example, the B. subtilis amino acid sequence forxylanase A is shown as SEQ I.D. No. 1. With respect to this sequence,the surface amino acid residues are:

[0025] Ala1-Trp6, Asn8, Thr10-Gly23, Asn25, Ser27, Asn29, Ser31-Asn32,Gly34, Thr43-Thr44, Ser46-Thr50, Asn52, Asn54, Gly56-Asn61, Asn63,Arg73-Leu76, Thr87-Arg89, Thr91-Lys95, Thr97, Lys99, Asp101-Gly102,Thr104, Thr109-Thr111, Tyr113-Asn114, Asp119-Thr124, Thr126,Gln133-Asn141, Thr143, Thr145, Thr147-Asn148, Asn151, Lys154-Gly157,Asn159-Leu160, Ser162-Trp164, Gln175, Ser177, Ser179, Asn181, Thr183,Trp185.

[0026] As indicated, the surface amino acids of other xylanase enzymes(such as Thermomyces lanuginosus xylanase A, whose coding nucleotidesequence is presented as SEQ ID No. 9) are determinable by a personskilled in the art.

[0027] Hence, for some aspects the present invention encompasses avariant xylanase polypeptide, or fragment thereof having xylanaseactivity, which variant xylanase polypeptide or fragment comprises oneor more amino acid modifications at any one of amino acid residues:

[0028] Ala1-Trp6, Asn8, Thr10-Gly23, Asn25, Ser27, Asn29, Ser31-Asn32,Gly34, Thr43-Thr44, Ser46-Thr50, Asn52, Asn54, Gly56-Asn61, Asn63,Arg73-Leu76, Thr87-Arg89, Thr91-Lys95, Thr97, Lys99, Asp101-Gly102,Thr104, Thr109-Thr111, Tyr113-Asn114, Asp119-Thr124, Thr126,Gln133-Asn141, Thr143, Thr145, Thr147-Asn148, Asn151, Lys154-Gly157,Asn159-Leu160, Ser162-Trp164, Gln175, Ser177, Ser179, Asn181, Thr183,Trp185

[0029] of the B. subtilis amino acid sequence shown as SEQ I.D. No. 1 orits/their equivalent positions in other homologous xylanasepolypeptides.

[0030] Thus, in one embodiment, the present invention provides a variantxylanase polypeptide, or fragment thereof having xylanase activity,comprising one or more amino acid modifications at any one of amino acidresidues numbers:

[0031] 11, 12, 13, 15, 17, 29, 31, 32, 34, 113, 114, 119, 120, 121, 122,123, 124 and 175

[0032] of the B. subtilis amino acid sequence shown as SEQ I.D. No. 1 ortheir equivalent positions in other homologous xylanase polypeptides.

[0033] In one embodiment, the present invention provides a variantxylanase polypeptide or fragment thereof having xylanase activity,comprising one or more amino acid modifications at any one of amino acidresidues numbers 11, 12 and 13 of the B. subtilis amino acid sequenceshown as SEQ I.D. No. 1 or their equivalent positions in otherhomologous xylanase polypeptides.

[0034] Specific preferred examples of modifications made are presentedin the Examples section herein.

[0035] For some embodiments, preferably the variant xylanasepolypeptide, or fragment thereof having xylanase activity, comprises oneor more amino acid modifications at any one of amino acid residuesnumbers: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 29, 30, 31, 32, 33,34, 35, 36, 37, 61, 62, 63, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 173, 174, 175, 176, 177, 178 of the B.subtilis amino acid sequence shown as SEQ I.D. No. 1 or their equivalentpositions in other homologous xylanase polypeptides.

[0036] For convenience, we sometimes refer to the amino acid residuesnumbers: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 29, 30, 31, 32, 33,34, 35, 36, 37, 61, 62, 63, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 173, 174, 175, 176, 177, 178 as BAND 1.

[0037]FIG. 1 shows the 3-D structure of B. subtilis xylanase having theamino acid sequence shown as SEQ I.D. No. 1. BAND 1 is depicted in FIG.1 as the upper layer of the molecule and extends approximately 13Angstroms from the top of the molecule when the molecule is orientatedas shown in FIG. 1. BAND 1 ends with the residue Phe 125 on the lefthand side when viewing FIG. 1 and with the residue Asn 61 on the righthand side when viewing FIG. 1.

[0038] In addition, or in the alternative, for some embodiments,preferably the variant xylanase polypeptide, or fragment thereof havingxylanase activity, comprises one or more amino acid modifications at anyone of the other amino acid residues.

[0039] Preferably said other modifications may occur at any one or moreof amino acid residues numbers: 3, 4, 5, 6, 7, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 38, 39, 40, 41, 42, 43, 44, 45, 55, 56, 57, 58, 59, 60,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 108, 109, 110, 126,127, 128, 129, 130, 131, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 179, 180, 181, 182, 183 of the B. subtilisamino acid sequence shown as SEQ I.D. No.1 or their equivalent positionsin other homologous xylanase polypeptides.

[0040] For convenience, we sometimes refer to the amino acid residuesnumbers: 3, 4, 5, 6, 7, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 38, 39,40, 41, 42, 43, 44, 45, 55, 56, 57, 58, 59, 60, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 108, 109, 110, 126, 127, 128, 129, 130, 131,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 179, 180, 181, 182, 183 of the B. subtilis amino acid sequenceshown as BAND 2.

[0041] Preferably said other modifications may occur at any one or moreof the surface amino acid residues numbers: 3, 4, 5, 6, 19, 20, 21, 22,23, 25, 27, 43, 44, 56, 57, 58, 59, 60, 73, 74, 75, 76, 87, 89, 91, 92,93, 94, 109, 110, 126, 159, 160, 162, 163, 164, 179, 181, 183 of the B.subtilis amino acid sequence shown as SEQ I.D. No.1 or their equivalentpositions in other homologous xylanase polypeptides.

[0042] Preferably, the present invention encompasses a variant xylanasepolypeptide, or fragment thereof having xylanase activity, whichcomprises one or more amino acid modifications in BAND 1 andoptionally/or BAND 2 of the B. subtilis amino acid sequence or theirequivalent positions (bands) in other homologous xylanase polypeptides.Hence, the modification is in at least BAND 1; but could be in just BAND2 alone.

[0043] The variant xylanase polypeptide may comprise other modificationsin other amino acid residues, such as modification at any one of aminoacid residues: 1, 2, 46, 47, 48, 49, 50, 51, 52, 53, 54, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 184, 185 of the B. subtilis aminoacid sequence shown as SEQ I.D. No. 1 or their equivalent positions inother homologous xylanase polypeptides.

[0044] The variant xylanase polypeptide may comprise other modificationsin other surface amino acid residues, such as modification at any one ofthe surface amino acid residues: 1, 2, 46, 47, 48, 49, 50, 52, 54, 95,97, 99, 101, 102, 104, 133, 134, 135, 136, 137, 138, 139, 140, 141, 143,145, 147, 148, 151, 154, 155, 156, 157, 185 of the B. subtilis aminoacid sequence shown as SEQ I.D. No. 1 or their equivalent positions inother homologous xylanase polypeptides.

[0045] Preferably, the inhibitor is an inhibitor found naturally inplant tissues. Preferably the sensitivity of the variant xylanase enzymeto the inhibitor is reduced as compared to the parent xylanase enzyme.

[0046] The present invention also provides a nucleic acid molecule (anucleotide sequence) encoding a polypeptide of the invention. Alsoprovided is a vector comprising a nucleic acid of the invention,optionally operably linked to a regulatory sequence capable of directingexpression of said nucleic acid in a suitable host cell. A host cellcomprising a nucleic acid or a vector of the invention is also provided.

[0047] In another aspect the present invention provides a method ofmaking a polypeptide of the invention comprising transforming a hostcell with a nucleic acid encoding said polypeptide, culturing thetransformed cell and expressing said polypeptide.

[0048] Our results show that these variant polypeptides have improvedproperties that make them suitable for a variety of applications, suchas bakery, animal feed, starch production, flour separation (wetmilling)and, paper and pulp production.

[0049] Accordingly, the present invention also provides the use of avariant polypeptide of the invention in a method of modifying plantmaterials.

[0050] Also provided is the use of a variant polypeptide of theinvention in baking. The invention further provides the use of a variantpolypeptide of the invention in processing cereals, starch productionand animal feed and the use of a variant polypeptide of the invention inprocessing wood, for example in enhancing the bleaching of wood pulp.

[0051] In a further aspect, the present invention provides a method ofaltering the sensitivity of a xylanase polypeptide to an inhibitor whichmethod comprises modifying one or more amino acid residues of saidenzyme selected from amino acid numbers 11, 12, 13, 15, 17, 29, 31, 32,34, 113, 114, 119, 120, 121, 122, 123, 124 and 175 based on the aminoacid numbering of B. subtilis xylanase shown as SEQ ID No. 1, or theequivalent residues in other homologous xylanase polypeptides.Preferably the sensitivity is reduced.

[0052] Importantly, our results also show for the first time thatxylanase inhibitors play an important role in determining thefunctionality of xylanase enzymes in a complex system, such as a plantmaterial. By the term “functionality”, we mean the biochemicalproperties of the xylanase in a given system. These properties includesubstrate specificity, K_(m) and V_(max) kinetic parameters (whereappropriate) and the nature of the reaction products obtained by theaction of the xylanase in that system. Functionality may alsoconsequently be described in terms of the effect on the physical and/orchemical properties of the plant materials on which the xylanase acts,for example the extent to which the viscosity of the material isaltered.

[0053] In the same way that variant xylanases may be used in a varietyof processing applications, xylanase inhibitors may be used in a varietyof processing applications such as bakery, wood pulp processing andcereal processing.

DETAILED DESCRIPTION OF THE INVENTION

[0054] Although in general any molecular techniques mentioned herein arewell known in the art, reference may be made in particular to Sambrooket al., Molecular Cloning, A Laboratory Manual (1989) and Ausubel etal., Short Protocols in Molecular Biology (1999)₄th Ed, John Wiley &Sons, Inc.

[0055] A. Variant Xylanase Polypeptides

[0056] Xylanase enzymes have been reported from nearly 100 differentorganisms, including plants, fungi and bacteria. The xylanase enzymesare classified into several of the more than 40 families of glycosylhydrolase enzymes. The glycosyl hydrolase enzymes, which includexylanases, mannanases, amylases, β-glucanases, cellulases, and othercarbohydrases, are classified based on such properties as the sequenceof amino acids, the three dimensional structure and the geometry of thecatalytic site (Gilkes, et al., 1991, Microbiol. Reviews 55: 303-315).

[0057] Of particular interest for baking applications are the enzymesclassified in Family 11. All of these are xylanases and are known as the“Family 11 xylanases”. Some publications refer to these synonymously asthe Family G xylanases, but the term “Family 11 xylanases” will be usedherein to refer to both Family G and Family 11 xylanases.

[0058] Table A lists a number of known Family 11 xylanases. Most of themhave a molecular mass of about 21,000 Da. Three of the Family 11xylanases (Clostridium stercorarium XynA, Streptomyces lividans XynB,and Thermomonospora fusca XynA) have a higher molecular mass of 31,000to 50,000 Da. However, these xylanases have a catalytic core sequence ofabout 21,000 Da similar to the other Family 11 xylanases. The amino acidsequences of the Family 11 xylanases (or, for the larger enzymes, thecatalytic core) show a high degree of similarity, usually with more than40% identical amino acids in a proper amino acid alignment. The Family11 xylanases, which are of bacterial, yeast, or fungal origin, share thesame general molecular structure.

[0059]FIG. 2 shows amino acid sequence alignment data in respect of 51Family 11 xylanases. TABLE A Family 11 xylanases Aspergillus niger Xyn AAspergillus kawachii Xyn C Aspergillus tubigensis Xyn A Bacilluscirculans Xyn A Bacillus pumilus Xyn A Bacillus subtilis Xyn ACellulomonas fimi Xyn D Chainia spp. Xyn Clostridium acetobutylicum XynB Clostridium stercorarium Xyn A Fibrobacter succinogenes Xyn CNeocallimastix patriciarum Xyn A Nocardiopsis dassonvillei Xyn IIRuminococcus flavefaciens Xyn A Schizophyllum commune Xyn Streptomyceslividans Xyn B Streptomyces lividans Xyn C Streptomyces sp. No. 36a XynStreptomyces thermoviolaceus Xyn II Thermomonospora fusca Xyn ATrichoderma harzianum Xyn Trichoderma reesei Xyn I Trichoderma reeseiXyn II Trichoderma viride Xyn

[0060] Variant Xylanases of the Invention

[0061] A variant xylanase polypeptide of the invention is typicallyobtained by modifying a xylanase polypeptide by substituting, deletingor adding one or more amino acid residues within the amino acid sequenceof the xylanase polypeptide. Preferably the modification comprises oneor more amino acid substitutions. Modification of polypeptide sequencescan be carried out using standard techniques such as site directedmutagenesis. The modification may also occur by chemical techniques—suchas chemical modification of one or more amino acid residues.

[0062] The starting sequence may be a wild type sequence or anon-naturally occurring sequence, for example a derivative that hasalready been subjected to protein engineering. The xylanase sequence tobe modified may be from any source, for example a bacterial, fungal orplant source. Preferably the xylanase sequence to be modified is that ofa Family 11 xylanase, more preferably a Family 11 xylanase selected fromTrichoderma reesei xylanase I, Trichoderma reesei xylanase II,Trichoderma harzianum xylanase, Trichoderma viride xylanase, Bacilluscirculans xylanase A, Bacillus subtilis xylanase A, Aspergillus nigerxylanase A, Aspergillus kawachii xylanase C, Aspergillus tubigensisxylanase A, Streptomyces lividans xylanase B, and Streptomyces lividansxylanase C.

[0063] In a particularly preferred embodiment, the xylanase sequence tobe modified is the B subtilis xylanase sequence shown as SEQ ID No. 1 ora homologue thereof. Preferably said homologue has at least 40, 50, 60or 80% homology over at least 50 or 100 amino acid residues asdetermined using the GCG Wisconsin Bestfit package (University ofWisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research12:387).

[0064] Specific modifications that are preferred according to thepresent invention include one or more amino acid substitutions atpositions 11, 12, 13, 15, 17, 29, 31, 32, 34, 113, 114, 119, 120, 121,122, 123, 124 and 175 based on the amino acid numbering of B. subtilisxylanase shown as SEQ ID No. 1, or the equivalent residues in otherhomologous xylanase polypeptides.

[0065] Particularly preferred substitutions include one or more ofD11→Y, D11→N, D11→F, D11→K, D11→S, D11→W, G12→F, G13→F, I15→K, N17→K,N17→Y, N17→D, N29→K, N29→Y, N29→D, S31→K, S31→Y, S31→D, N32→K, G34→D,G34→F, G34→T, Y113→A, Y113→D, Y113→K, N114→A, N114→D, N114→F, N114→K,D119→K, D119→Y, D119→N, G120→K, G120→D, G120→F, G120→Y, G120→N, D121→N,D121→K, D121→F, D121→A, R122→D, R122→F, R122→A, T123→K, T123→Y, T123→D,T124→K, T124→Y, T124→D, Q175→E, Q175→S and Q175→L (with reference to theamino acid sequence of B. subtilis xylanase) or their equivalents inother homologous xylanase polypeptides. Further references to specificresidues of the B. subtilis xylanase shown as SEQ ID No. 1 will alsoinclude their equivalents in other homologous xylanase polypeptides.

[0066] A combination of mutations may be carried out, for examplemutations at two or more of the above-mentioned residues. Examples ofsuch combinations are presented in the Examples section herein.

[0067] In a further embodiment, the variant polypeptides of theinvention may be purified and isolated naturally occurring mutantxylanases. Alternatively, mutant xylanases may be generated bysubjecting organisms to mutagens and then screening for individualscomprising mutations in their xylanase genes. Naturally occurringmutants and mutants generated by random mutagenesis may beidentified/screened using a variety of techniques such as PCR screeningusing suitable nucleic acid primers to amplify regions of xylanase genesand sequencing the resulting fragments.

[0068] Thus variant polypeptides of the invention include naturallyoccurring mutant xylanases (purified and isolated from the organisms inwhich they occur or obtained recombinantly), mutant xylanases obtainedby random mutagenesis and mutant xylanases obtained by site-directedmutagenesis.

[0069] Variant polypeptides of the invention may also be subjected tofurther modifications that do not necessarily affect sensitivity toinhibitors, including any substitution of, variation of, modificationof, replacement of, deletion of or addition of one (or more) amino acidsfrom or to the sequence providing the resultant amino acid sequenceretains xylanase activity, preferably having at least substantially thesame xylanase activity as the unmodified sequence.

[0070] Conservative substitutions may be made, for example according tothe Table below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other: ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M NQ Polar - charged D E K R AROMATIC H F W Y

[0071] Polypeptides of the invention also include fragments of the fulllength sequences mentioned above having xylanase activity.

[0072] Polypeptides of the invention may further comprise heterologousamino acid sequences, typically at the N-terminus or C-terminus,preferably the N-terminus. Heterologous sequence may include sequencesthat affect intra or extracellular protein targeting (such as leadersequences).

[0073] Polypeptides of the invention are typically made by recombinantmeans, for example as described below. However they may also be made bysynthetic means using techniques well known to skilled persons such assolid phase synthesis. Polypeptides of the invention may also beproduced as fusion proteins, for example to aid in extraction andpurification. It may also be convenient to include a proteolyticcleavage site between the fusion protein partner and the proteinsequence of interest to allow removal of fusion protein sequences, suchas a thrombin cleavage site. Preferably the fusion protein will nothinder the function of the protein sequence of interest.

[0074] The use of appropriate host cells is expected to provide for suchpost-translational modifications as may be needed to confer optimalbiological activity on recombinant expression products of the invention.

[0075] Polypeptides of the invention may be in a substantially isolatedform. It will be understood that the protein may be mixed with carriersor diluents which will not interfere with the intended purpose of theprotein and still be regarded as substantially isolated. A polypeptideof the invention may also be in a substantially purified form, in whichcase it will generally comprise the protein in a preparation in whichmore than 90%, e.g. 95%, 98% or 99% of the protein in the preparation isa polypeptide of the invention.

[0076] Variant polypeptides of the invention have altered sensitivity toxylanase inhibitors compared to the parent xylanase sequence—which maybe a corresponding wild type xylanase. Preferably, variant polypeptideshave reduced sensitivity to xylanase inhibitors. The term “alteredsensitivity to xylanase inhibitors” means that extent to which theendo-β-1,4-xylanase activity of a variant polypeptide of the inventionis inhibited by the xylanase inhibitor is different to that of theparent xylanase enzyme—which may be the corresponding wild typexylanase. Preferably the extent to which the variant polypeptide isinhibited by the inhibitor is less than that of the parent xylanaseenzyme—which may be the wild type protein. This may, for example, be dueto a change in the three-dimensional structure of the variantpolypeptide such that the inhibitor no longer binds with the sameaffinity as it does to the parent xylanase enzyme—which may be the wildtype enzyme.

[0077] The sensitivity of the variant polypeptides of the invention toxylanase inhibitors can be assayed using, for example, the assaydescribed in example 4 and below. A suitable inhibitor for use in theassay is the inhibitor purified from wheat flour in example 1. Otherinhibitors are described below.

[0078] Xylanase Assay (Endo-β-1,4-Xylanase Activity)

[0079] Xylanase samples are diluted in citric acid (0.1M)-di-sodium-hydrogen phosphate (0.2 M) buffer, pH 5.0, to obtainapprox. OD=0.7 in the final assay. Three dilutions of the sample and aninternal standard with a defined activity are thermostated for 5 minutesat 40° C. At time=5 minutes, 1 Xylazyme tab (crosslinked, dyed xylansubstrate) is added to the enzyme solution. At time=15 minutes (or insome cases longer, depending on the xylanase activity present in thesample) the reaction is terminated, by adding 10 ml of 2% TRIS. Thereaction mixture is centrifuged and the OD of the supernatant ismeasured at 590 nm. Taking into account the dilutions and the amount ofxylanase, the activity (TXU, Total-Xylanase-Units) of the sample can becalculated relative to the standard.

[0080] Xylanase Inhibitors

[0081] As used herein, the term “xylanase inhibitor” refers to acompound, typically a protein, whose role is to control thedepolymerisation of complex carbohydrates, such as arabinoxylan, foundin plant cell walls. These xylanase inhibitors are capable of reducingthe activity of naturally occurring xylanase enzymes as well as those offungal or bacterial origin. Although the presence of xylanase inhibitorshave been reported in cereal seeds (see for example McLauchlan et al1999a; Rouau and Suget 1998) their impact on the efficacy of xylanaseenzymes has not been extensively examined.

[0082] McLauchlan et al (1999a) disclose the isolation andcharacterisation of a protein from wheat that binds to and inhibits twofamily-11 xylanases. Likewise, WO 98/49278 demonstrates the effect of awheat flour extract on the activity of a group of microbial xylanasesall of which are classified as family 11 xylanases. Debyser et al.(1999) also disclose that endoxylanases from Aspergillus niger andBacillus subtilis, which are both members of the family 11 xylanaseswere inhibited by a wheat xylanase inhibitor called TAXI. McLauchlan etal (1999b) teach that extracts from commercial flours such as wheat,barley, rye and maize are capable of inhibiting both family 10 and 11xylanases.

[0083] The xylanase inhibitor may be any suitable xylanase inhibitor. Byway of example, the xylanase inhibitor may be the inhibitor described inWO-A-98/49278 and/or the xylanase inhibitor described by Rouau, X. andSurget, A. (1998), McLauchlan, R., et al. (1999) and/or the xylanaseinhibitor described in UK patent application number 9828599.2 (filedDec. 23, 1998), UK patent application number 9907805.7 (filed Apr. 6,1999) and UK patent application number 9908645.6 (filed Apr. 15, 1999).

[0084] Xylanase Inhibitor Assay

[0085] 100 μl of an candidate inhibitor fraction, 250 μl xylanasesolution (containing 12 TXU microbial xylanase/ml) and 650 μl buffer(0.1 M citric acid-0.2M di-sodium hydrogen phosphate buffer, pH 5.0) aremixed. The mixture is thermostated for 5 minutes at 40.0° C. At time=5minutes one Xylazyme tab is added. At time=15 minutes the reaction isterminated by adding 10 ml 2% TRIS. The reaction mixture is centrifuged(3500 g, 10 minutes, room temperature) and the supernatant is measuredat 590 nm. The inhibition is calculated as residual activity compared tothe blank. The blank is prepared the same way, except that the 100 μlinhibitor is substituted with 100 μl buffer (0.1 M citric acid-0.2 Mdi-sodium hydrogen phosphate buffer, pH 5.0).

[0086] Specific Xylanase Inhibitor

[0087] As indicated, a xylanase inhibitor that may be used in accordancewith the present invention is the xylanase inhibitor described in UKpatent application number 9828599.2 (filed Dec. 23, 1998), UK patentapplication number 9907805.7 (filed Apr. 6, 1999) and UK patentapplication number 9908645.6 (filed Apr. 15, 1999).

[0088] This endogenous endo-β-1,4-xylanase inhibitor is obtainable fromwheat flour. The inhibitor is a di-peptide, having a MW of about 40 kDa(as measured by SDS-PAGE or mass spectrometry) and a pI of about 8 toabout 9.5.

[0089] Sequence analysis to date has revealed the that the inhibitor hasat least one or more of the sequences presented as SEQ ID No. 2, SEQ IDNo. 3, SEQ ID No 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 and/or SEQID No. 8.

[0090] These inhibitors described in the prior art may also be used inassays to determine the sensitivity of a variant polypeptide of theinvention to xylanase inhibitors. They may also be used as describedbelow to modulate the functionality of a xylanase.

[0091] Polynucleotides

[0092] Polynucleotides of the invention comprise nucleic acid sequencesencoding the variant polypeptide sequences of the invention. It will beunderstood by a skilled person that numerous different polynucleotidescan encode the same polypeptide as a result of the degeneracy of thegenetic code. In addition, it is to be understood that skilled personsmay, using routine techniques, make nucleotide substitutions that do notaffect the polypeptide sequence encoded by the polynucleotides of theinvention to reflect the codon usage of any particular host organism inwhich the polypeptides of the invention are to be expressed.

[0093] Polynucleotides of the invention may comprise DNA or RNA. Theymay be single-stranded or double-stranded. They may also bepolynucleotides which include within them synthetic or modifiednucleotides. A number of different types of modification tooligonucleotides are known in the art. These include methylphosphonateand phosphorothioate backbones, addition of acridine or polylysinechains at the 3′ and/or 5′ ends of the molecule. For the purposes of thepresent invention, it is to be understood that the polynucleotidesdescribed herein may be modified by any method available in the art.Such modifications may be carried out in order to enhance the in vivoactivity or life span of polynucleotides of the invention.

[0094] Nucleotide Vectors and Host Cells

[0095] Polynucleotides of the invention can be incorporated into arecombinant replicable vector. The vector may be used to replicate thenucleic acid in a compatible host cell. Thus in a further embodiment,the invention provides a method of making polynucleotides of theinvention by introducing a polynucleotide of the invention into areplicable vector, introducing the vector into a compatible host cell,and growing the host cell under conditions which bring about replicationof the vector. The vector may be recovered from the host cell. Suitablehost cells include bacteria such as E. coli, yeast and fungi.

[0096] Preferably, a polynucleotide of the invention in a vector isoperably linked to a regulatory sequence which is capable of providingfor the expression of the coding sequence by the host cell, i.e. thevector is an expression vector. The term “operably linked” refers to ajuxtaposition wherein the components described are in a relationshippermitting them to function in their intended manner. A regulatorysequence “operably linked” to a coding sequence is ligated in such a waythat expression of the coding sequence is achieved under conditionscompatible with the control sequences. The term “regulatory sequences”includes promoters and enhancers and other expression regulationsignals.

[0097] Enhanced expression of the polynucleotide encoding thepolypeptide of the invention may also be achieved by the selection ofheterologous regulatory regions, e.g. promoter, secretion leader andterminator regions, which serve to increase expression and, if desired,secretion levels of the protein of interest from the chosen expressionhost and/or to provide for the inducible control of the expression ofthe polypeptide of the invention.

[0098] Aside from the promoter native to the gene encoding thepolypeptide of the invention, other promoters may be used to directexpression of the polypeptide of the invention. The promoter may beselected for its efficiency in directing the expression of thepolypeptide of the invention in the desired expression host.

[0099] In another embodiment, a constitutive promoter may be selected todirect the expression of the desired polypeptide of the invention.Examples of strong constitutive and/or inducible promoters which arepreferred for use in fungal expression hosts are those which areobtainable from the fungal genes for xylanase (xlnA), phytase,ATP-synthetase, subunit 9 (oliC), triose phosphate isomerase (tpi),alcohol dehydrogenase (AdhA), α-amylase (amy), amyloglucosidase (AG-fromthe glaA gene), acetamidase (amdS) and glyceraldehyde-3-phosphatedehydrogenase (gpd) promoters.

[0100] Examples of strong yeast promoters are those obtainable from thegenes for alcohol dehydrogenase, lactase, 3-phosphoglycerate kinase andtriosephosphate isomerase.

[0101] Examples of strong bacterial promoters are the α-amylase and SPO₂promoters as well as promoters from extracellular protease genes.

[0102] Hybrid promoters may also be used to improve inducible regulationof the expression construct.

[0103] Often, it is desirable for the polypeptide of the invention to besecreted from the expression host into the culture medium from where thepolypeptide of the invention may be more easily recovered. According tothe present invention, the polypeptide of the invention's nativesecretion leader sequence may be used to effect the secretion of theexpressed polypeptide of the invention. However, an increase in theexpression of the polypeptide of the invention sometimes results in theproduction of the protein in levels beyond that which the expressionhost is capable of processing and secreting, creating a bottleneck suchthat the protein product accumulates within the cell. Accordingly, thepresent invention also provides heterologous leader sequences to providefor the most efficient secretion of the polypeptide of the inventionfrom the chosen expression host.

[0104] According to the present invention, the secretion leader may beselected on the basis of the desired expression host. A heterologoussecretion leader may be chosen which is homologous to the otherregulatory regions of the expression construct. For example, the leaderof the highly secreted amyloglucosidase (AG) protein may be used incombination with the amyloglucosidase (AG) promoter itself, as well asin combination with other promoters. Hybrid signal sequences may also beused with the context of the present invention. Examples of preferredheterologous secretion leader sequences are those originating from thefungal amyloglucosidase (AG) gene (glaA—both 18 and 24 amino acidversions e.g. from Aspergillus), the α-factor gene (yeasts e.g.Saccharomyces and Kluyveromyces) or the α-amylase gene (Bacillus).

[0105] Such vectors may be transformed into a suitable host cell asdescribed above to provide for expression of a polypeptide of theinvention. Thus, in a further aspect the invention provides a processfor preparing polypeptides according to the invention which comprisescultivating a host cell transformed or transfected with an expressionvector as described above under conditions to provide for expression bythe vector of a coding sequence encoding the polypeptides, andrecovering the expressed polypeptides. Suitable host cells include, forexample, fungal cells, such as Aspergillus and yeast cells, such asyeast cells of the genus Kluyveromyces or Saccharomyces. Other suitablehost cells are discussed below.

[0106] The vectors may be for example, plasmid, virus or phage vectorsprovided with an origin of replication, optionally a promoter for theexpression of the said polynucleotide and optionally a regulator of thepromoter. The vectors may contain one or more selectable marker genes.The most suitable selection systems for industrial micro-organisms arethose formed by the group of selection markers which do not require amutation in the host organism. Examples of fungal selection markers arethe genes for acetamidase (amdS), ATP synthetase, subunit 9 (oliC),orotidine-5′-phosphate-decarboxylase (pvrA), phleomycin and benomylresistance (benA). Examples of non-fungal selection markers are thebacterial G418 resistance gene (this may also be used in yeast, but notin fungi), the ampicillin resistance gene (E. coli), the neomycinresistance gene (Bacillus) and the E. coli uidA gene, coding forβ-glucuronidase (GUS). Vectors may be used in vitro, for example for theproduction of RNA or used to transfect or transform a host cell.

[0107] A further embodiment of the invention provides host cellstransformed or transfected with a polynucleotide of the invention.Preferably said polynucleotide is carried in a vector for thereplication and expression of said polynucleotides. The cells will bechosen to be compatible with the said vector and may for example beprokaryotic (for example bacterial), fungal, yeast or plant cells.

[0108] Bacteria from the genus Bacillus are very suitable asheterologous hosts because of their capability to secrete proteins intothe culture medium. Other bacteria suitable as hosts are those from thegenera Streptomyces and Pseudomonas.

[0109] Depending on the nature of the polynucleotide encoding thepolypeptide of the invention, and/or the desirability for furtherprocessing of the expressed protein, eukaryotic hosts such as yeasts orfungi may be preferred. In general, yeast cells are preferred overfungal cells because they are easier to manipulate. However, someproteins are either poorly secreted from the yeast cell, or in somecases are not processed properly (e.g. hyperglycosylation in yeast). Inthese instances, a fungal host organism should be selected.

[0110] A heterologous host may also be chosen wherein the polypeptide ofthe invention is produced in a form which is substantially free fromother xylanases. This may be achieved by choosing a host which does notnormally produce such enzymes.

[0111] Examples of preferred expression hosts within the scope of thepresent invention are fungi such as Aspergillus species and Trichodermaspecies; bacteria such as Bacillus species, Streptomyces species andPseudomonas species; and yeasts such as Kluyveromyces species andSaccharomyces species.

[0112] Particularly preferred expression hosts may be selected fromAspergillus niger, Aspergillus niger var. tubigenis, Aspergillus nigervar. awamori, Aspergillus aculeatis, Aspergillus nidulans, Aspergillusoryzae, Trichoderma reesei, Bacillus subtilis, Bacillus licheniformis,Bacillus amyloliquefaciens, Kluyveromyces lactis and Saccharomycescerevisiae.

[0113] According to the present invention, the production of thepolypeptide of the invention can be effected by the culturing ofmicrobial expression hosts, which have been transformed with one or morepolynucleotides of the present invention, in a conventional nutrientfermentation medium.

[0114] The fermentation medium can comprise a known culture mediumcontaining a carbon source (e.g. glucose, maltose, molasses, etc.), anitrogen source (e.g. ammonium sulphate, ammonium nitrate, ammoniumchloride, etc.), an organic nitrogen source (e.g. yeast extract, maltextract, peptone, etc.) and inorganic nutrient sources (e.g. phosphate,magnesium, potassium, zinc, iron, etc.). Optionally, an inducer may beadded.

[0115] The selection of the appropriate medium may be based on thechoice of expression hosts and/or based on the regulatory requirementsof the expression construct. Such media are well-known to those skilledin the art. The medium may, if desired, contain additional componentsfavouring the transformed expression hosts over other potentiallycontaminating microorganisms.

[0116] After fermentation, the cells can be removed from thefermentation broth by means of centrifugation or filtration. Afterremoval of the cells, the variant polypeptide of the invention may thenbe recovered and, if desired, purified and isolated by conventionalmeans.

[0117] Organisms

[0118] The term “organism” in relation to the present invention includesany organism that could comprise the nucleotide sequence coding for thevariant xylanase protein according to the present invention and/orproducts obtained therefrom, wherein a transcriptional regulatorysequence can allow expression of the nucleotide sequence according tothe present invention when present in the organism. Suitable organismsmay include a prokaryote, fungus, yeast or a plant. For the xylanaseaspect of the present invention, a preferable organism may be abacterium, preferably of the genus Bacillus, more preferably Bacillussubtilis.

[0119] The term “transgenic organism” in relation to the presentinvention includes any organism that comprises the nucleotide sequencecoding for the protein according to the present invention and/orproducts obtained therefrom, wherein the transcriptional regulatorysequence can allow expression of the nucleotide sequence according tothe present invention within the organism. Preferably the nucleotidesequence is incorporated in the genome of the organism.

[0120] The term “transgenic organism” does not cover native nucleotidecoding sequences in their natural environment when they are under thecontrol of their native promoter which is also in its naturalenvironment.

[0121] Therefore, the transgenic organism of the present inventionincludes an organism comprising any one of, or combinations of, thenucleotide sequence coding for the amino acid sequence according to thepresent invention, constructs according to the present invention(including combinations thereof), vectors according to the presentinvention, plasmids according to the present invention, cells accordingto the present invention, tissues according to the present invention orthe products thereof. The transformed cell or organism could prepareacceptable quantities of the desired compound which would be easilyretrievable from, the cell or organism.

[0122] Transformation of Host Cells/Host Organisms

[0123] As indicated earlier, the host organism can be a prokaryotic or aeukaryotic organism. Examples of suitable prokaryotic hosts include E.coli and Bacillus subtilis. Teachings on the transformation ofprokaryotic hosts is well documented in the art, for example seeSambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition,1989, Cold Spring Harbor Laboratory Press) and Ausubel et al., ShortProtocols in Molecular Biology (1999), 4^(th) Ed., John Wiley & Sons,Inc.

[0124] If a prokaryotic host is used then the nucleotide sequence mayneed to be suitably modified before transformation—such as by removal ofintrons.

[0125] As mentioned above, a preferred host organism is of the genusBacillus, such as Bacillus subtilis.

[0126] In another embodiment the transgenic organism can be a yeast. Inthis regard, yeasts have also been widely used as a vehicle forheterologous gene expression. The species Saccharomyces cerevisiae has along history of industrial use, including its use for heterologous geneexpression. Expression of heterologous genes in Saccharomyces cerevisiaehas been reviewed by Goodey et al (1987, Yeast Biotechnology, D R Berryet al, eds, pp 401-429, Allen and Unwin, London) and by King et al(1989, Molecular and Cell Biology of Yeasts, E F Walton and G TYarronton, eds, pp 107-133, Blackie, Glasgow).

[0127] For several reasons Saccharomyces cerevisiae is well suited forheterologous gene expression. First, it is non-pathogenic to humans andit is incapable of producing certain endotoxins. Second, it has a longhistory of safe use following centuries of commercial exploitation forvarious purposes. This has led to wide public acceptability. Third, theextensive commercial use and research devoted to the organism hasresulted in a wealth of knowledge about the genetics and physiology aswell as large-scale fermentation characteristics of Saccharomycescerevisiae.

[0128] A review of the principles of heterologous gene expression inSaccharomyces cerevisiae and secretion of gene products is given by EHinchcliffe E Kenny (1993, “Yeast as a vehicle for the expression ofheterologous genes”, Yeasts, Vol 5, Anthony H Rose and J StuartHarrison, eds, 2nd edition, Academic Press Ltd.).

[0129] Several types of yeast vectors are available, includingintegrative vectors, which require recombination with the host genomefor their maintenance, and autonomously replicating plasmid vectors.

[0130] In order to prepare the transgenic Saccharomyces, expressionconstructs are prepared by inserting the nucleotide sequence of thepresent invention into a construct designed for expression in yeast.Several types of constructs used for heterologous expression have beendeveloped. The constructs contain a promoter active in yeast fused tothe nucleotide sequence of the present invention, usually a promoter ofyeast origin, such as the GAL1 promoter, is used. Usually a signalsequence of yeast origin, such as the sequence encoding the SUC2 signalpeptide, is used. A terminator active in yeast ends the expressionsystem.

[0131] For the transformation of yeast several transformation protocolshave been developed. For example, a transgenic Saccharomyces accordingto the present invention can be prepared by following the teachings ofHinnen et al (1978, Proceedings of the National Academy of Sciences ofthe USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito,H et al (1983, J Bacteriology 153, 163-168).

[0132] The transformed yeast cells are selected using various selectivemarkers. Among the markers used for transformation are a number ofauxotrophic markers such as LEU2, HIS4 and TRP1, and dominant antibioticresistance markers such as aminoglycoside antibiotic markers, e.g. G418.

[0133] Another host organism is a plant. The basic principle in theconstruction of genetically modified plants is to insert geneticinformation in the plant genome so as to obtain a stable maintenance ofthe inserted genetic material.

[0134] A transgenic plant of the invention may be produced from anyplant such as the seed-bearing plants (angiosperms), and conifers.Angiosperms include dicotyledons and monocotyledons. Examples ofdicotyledonous plants include tobacco, (Nicotiana plumbaginifolia andNicotiana tabacum), arabidopsis (Arabidopsis thaliana), Brassica napus,Brassica nigra, Datura innoxia, Vicia narbonensis, Vicia faba, pea(Pisum sativum), cauliflower, carnation and lentil (Lens culinaris).Examples of monocotyledonous plants include cereals such as wheat,barley, oats and maize.

[0135] Techniques for producing transgenic plants are well known in theart. Typically, either whole plants, cells or protoplasts may betransformed with a suitable nucleic acid construct encoding a zincfinger molecule or target DNA (see above for examples of nucleic acidconstructs). There are many methods for introducing transforming DNAconstructs into cells, but not all are suitable for delivering DNA toplant cells. Suitable methods include Agrobacterium infection (see,among others, Turpen et al., 1993, J. Virol. Methods, 42: 227-239) ordirect delivery of DNA such as, for example, by PEG-mediatedtransformation, by electroporation or by acceleration of DNA coatedparticles. Acceleration methods are generally preferred and include, forexample, microprojectile bombardment. A typical protocol for producingtransgenic plants (in particular monocotyledons), taken from U.S. Pat.No. 5, 874, 265, is described below.

[0136] An example of a method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method,non-biological particles may be coated with nucleic acids and deliveredinto cells by a propelling force. Exemplary particles include thosecomprised of tungsten, gold, platinum, and the like.

[0137] A particular advantage of microprojectile bombardment, inaddition to it being an effective means of reproducibly stablytransforming both dicotyledons and monocotyledons, is that neither theisolation of protoplasts nor the susceptibility to Agrobacteriuminfection is required. An illustrative embodiment of a method fordelivering DNA into plant cells by acceleration is a Biolistics ParticleDelivery System, which can be used to propel particles coated with DNAthrough a screen, such as a stainless steel or Nytex screen, onto afilter surface covered with plant cells cultured in suspension. Thescreen disperses the tungsten-DNA particles so that they are notdelivered to the recipient cells in large aggregates. It is believedthat without a screen intervening between the projectile apparatus andthe cells to be bombarded, the projectiles aggregate and may be toolarge for attaining a high frequency of transformation. This may be dueto damage inflicted on the recipient cells by projectiles that are toolarge.

[0138] For the bombardment, cells in suspension are preferablyconcentrated on filters. Filters containing the cells to be bombardedare positioned at an appropriate distance below the macroprojectilestopping plate. If desired, one or more screens are also positionedbetween the gun and the cells to be bombarded. Through the use oftechniques set forth herein one may obtain up to 1000 or more clustersof cells transiently expressing a marker gene (“foci”) on the bombardedfilter. The number of cells in a focus which express the exogenous geneproduct 48 hours post-bombardment often range from 1 to 10 and average 2to 3.

[0139] After effecting delivery of exogenous DNA to recipient cells byany of the methods discussed above, a preferred step is to identify thetransformed cells for further culturing and plant regeneration. Thisstep may include assaying cultures directly for a screenable trait or byexposing the bombarded cultures to a selective agent or agents.

[0140] An example of a screenable marker trait is the red pigmentproduced under the control of the R-locus in maize. This pigment may bedetected by culturing cells on a solid support containing nutrient mediacapable of supporting growth at this stage, incubating the cells at,e.g., 18° C. and greater than 180 μm⁻²s⁻¹, and selecting cells fromcolonies (visible aggregates of cells) that are pigmented. These cellsmay be cultured further, either in suspension or on solid media.

[0141] An exemplary embodiment of methods for identifying transformedcells involves exposing the bombarded cultures to a selective agent,such as a metabolic inhibitor, an antibiotic, herbicide or the like.Cells which have been transformed and have stably integrated a markergene conferring resistance to the selective agent used, will grow anddivide in culture. Sensitive cells will not be amenable to furtherculturing.

[0142] To use the bar-bialaphos selective system, bombarded cells onfilters are resuspended in nonselective liquid medium, cultured (e.g.for one to two weeks) and transferred to filters overlaying solid mediumcontaining from 1-3 mg/l bialaphos. While ranges of 1-3 mg/l willtypically be preferred, it is proposed that ranges of 0.1-50 mg/l willfind utility in the practice of the invention. The type of filter foruse in bombardment is not believed to be particularly crucial, and cancomprise any solid, porous, inert support.

[0143] Cells that survive the exposure to the selective agent may becultured in media that supports regeneration of plants. Tissue ismaintained on a basic media with hormones for about 2-4 weeks, thentransferred to media with no hormones. After 2-4 weeks, shootdevelopment will signal the time to transfer to another media.

[0144] Regeneration typically requires a progression of media whosecomposition has been modified to provide the appropriate nutrients andhormonal signals during sequential developmental stages from thetransformed callus to the more mature plant. Developing plantlets aretransferred to soil, and hardened, e.g., in an environmentallycontrolled chamber at about 85% relative humidity, 600 ppm CO₂, and 250μE m⁻² s⁻¹ of light. Plants are preferably matured either in a growthchamber or greenhouse. Regeneration will typically take about 3-12weeks. During regeneration, cells are grown on solid media in tissueculture vessels. An illustrative embodiment of such a vessel is a petridish. Regenerating plants are preferably grown at about 19° C. to 28° C.After the regenerating plants have reached the stage of shoot and rootdevelopment, they may be transferred to a greenhouse for further growthand testing.

[0145] Genomic DNA may be isolated from callus cell lines and plants todetermine the presence of the exogenous gene through the use oftechniques well known to those skilled in the art such as PCR and/orSouthern blotting.

[0146] Several techniques exist for inserting the genetic information,the two main principles being direct introduction of the geneticinformation and introduction of the genetic information by use of avector system. A review of the general techniques may be found inarticles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991]42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 199417-27).

[0147] Thus, in one aspect, the present invention relates to a vectorsystem which carries a construct encoding a variant xylanase polypeptideaccording to the present invention and which is capable of introducingthe construct into the genome of a plant.

[0148] The vector system may comprise one vector, but it can comprise atleast two vectors. In the case of two vectors, the vector system isnormally referred to as a binary vector system. Binary vector systemsare described in further detail in Gynheung An et al. (1980), BinaryVectors, Plant Molecular Biology Manual A3, 1-19.

[0149] One extensively employed system for transformation of plant cellswith a given promoter or nucleotide sequence or construct is based onthe use of a Ti plasmid from Agrobacterium tumefaciens or a R1 plasmidfrom Agrobacterium rhizogenes (An et al. (1986), Plant Physiol. 81,301-305 and Butcher D. N. et al. (1980), Tissue Culture Methods forPlant Pathologists, eds.: D. S. Ingrams and J. P. Helgeson, 203-208).

[0150] Several different Ti and Ri plasmids have been constructed whichare suitable for the construction of the plant or plant cell constructsdescribed above.

[0151] B. Uses

[0152] In a general sense, a variant xylanase of the invention may beused to alter, for example reduce, the viscosity derived from thepresence of hemicellulose or arabinoxylan in a solution or systemcomprising plant cell wall material. Typically said plant cell wallmaterials will comprise one or more xylanase inhibitors.

[0153] Specifically, a variant xylanase of the invention may be used inprocessing plant materials for use as foodstuffs, such as animal feed,in starch production, in baking and in the processing of wood pulp tomake paper.

[0154] Preparation of Foodstuffs

[0155] A variant xylanase of the invention may be used to process plantmaterials such as cereals that are used in foodstuffs including animalfeed. As used herein, the term “cereal” means any kind of grain used forfood and/or any grass producing this grain such as but not limited toany one of wheat, milled wheat, barley, maize, sorghum, rye, oats,triticale and rice or combinations thereof. In one preferred embodiment,the cereal is a wheat cereal.

[0156] The xylan in the food and/or feed supplement is modified bycontacting the xylan with the variant xylanase of the present invention.

[0157] As used herein, the term “contacting” includes but is not limitedto spraying, coating, impregnating or layering the food and/or feedsupplement with the variant xylanase enzyme of the present invention.

[0158] In one embodiment, the food and/or feed supplement of the presentinvention may be prepared by mixing the variant xylanase enzyme directlywith a food and/or feed supplement. By way of example, the variantxylanase enzyme may be contacted (for example, by spraying) onto acereal-based food and/or feed supplement such as milled wheat, maize orsoya flour.

[0159] It is also possible to incorporating the variant xylanase enzymeit into a second (and different) food and/or feed or drinking waterwhich is then added to the food and/or feed supplement of the presentinvention. Accordingly, it is not essential that the variant xylanaseenzyme provided by the present invention is incorporated into thecereal-based food and/or feed supplement itself, although suchincorporation forms a particularly preferred aspect of the presentinvention.

[0160] In one embodiment of the present invention, the food and/or feedsupplement may be combined with other food and/or feed components toproduce a cereal-based food and/or feed. Such other food and/or feedcomponents may include one or more other (preferably thermostable)enzyme supplements, vitamin food and/or feed supplements, mineral foodand/or feed supplements and amino acid food and/or feed supplements. Theresulting (combined) food and/or feed supplement comprising possiblyseveral different types of compounds can then be mixed in an appropriateamount with the other food and/or feed components such as cereal andprotein supplements to form a human food and/or an animal feed.

[0161] In one preferred embodiment, the food and/or feed supplement ofthe present invention can be prepared by mixing different enzymes havingthe appropriate activities to produce an enzyme mix. By way of example,a cereal-based food and/or feed supplement formed from e.g. milled wheator maize may be contacted (e.g. by spraying) either simultaneously orsequentially with the xylanase enzyme and other enzymes havingappropriate activities. These enzymes may include but are not limited toany one or more of an amylase, a glucoamylase, a mannanase, an agalactosidase, a phytase, a lipase, a glucanase, an-arabinofuranosidase,a pectinase, a protease, a glucose oxidase, a hexose oxidase and axylanase. Enzymes having the desired activities may for instance bemixed with the xylanase of the present invention either beforecontacting these enzymes with a cereal-based food and/or feed supplementor alternatively such enzymes may be contacted simultaneously orsequentially on such a cereal based supplement. The food and/or feedsupplement is then in turn mixed with a cereal-based food and/or feed toprepare the final food and/or feed. It is also possible to formulate thefood and/or feed supplement as a solution of the individual enzymeactivities and then mix this solution with a food and/or feed materialprior to processing the food and/or feed supplement into pellets or as amash.

[0162] Bakery Products

[0163] The present invention provides the use of a variant xylanasepolypeptide of the invention in a process for preparing a foodstuff.Typical bakery (baked) products in accordance with the present inventioninclude bread—such as loaves, rolls, buns, pizza bases etc.—pretzels,tortillas, cakes, cookies, biscuits, crackers etc. The preparation offoodstuffs such as bakery products is well know in the art. Doughproduction, for example, is described in example 2. The use of variantxylanases of the invention to alter the viscosity of a flour slurry indescribed in the example 5.

[0164] Starch Production

[0165] A variant xylanase of the invention may also be used in starchproduction from plant materials derived from cereals and tubers, such aspotatoes.

[0166] Processing of Wood Pulp

[0167] A variant xylanase of the invention may also be used inprocessing wood pulp, for example in the preparation of paper.

[0168] As discussed above, we have shown that a major determinant ofxylanase functionality is the presence of endogenous inhibitors in plantmaterial. Consequently, although one method for altering xylanasefunctionality is to modify a xylanase to change its sensitivity toendogenous inhibitors, another method would be to vary the amount and/ortype of inhibitor present in the plant material. Thus, the presentinvention also provides the use of a xylanase inhibitor to alter thefunctionality of a xylanase and consequently the use of a xylanaseinhibitor in the methods of processing plant materials described above.The present invention will now be further described with reference tothe following examples which are intended to be illustrative only andnon-limiting.

EXAMPLES Example 1 Purification and Characterisation of Wheat EndogenousXylanase Inhibitor

[0169] 2 kg wheat flour (Danish reform, batch 99056) was extracted withwater, using a flour:water ratio of 1:2, during 10 minutes of stirring.The soluble endogenous xylanase inhibitor was separated from theflour-water slurry by centrifugation. The extraction and centrifugationwas performed at 4° C. The inhibitor was purified from the water extractby the following chromatographic techniques and concentrationtechniques: HPLC-SEC, HPLC-CIEC, rotary evaporation, HPLC-HIC, HPLC-SECand rotary evaporation. The xylanase inhibitor could be monitored andquantified during purification, using the following quantificationmethod.

[0170] Inhibitor Quantification Method

[0171] 1 XIU (Xylanase Inhibitor Unit) is defined as the amount ofinhibitor that decreases 1 TXU to 0.5 TXU under the conditions describedbelow.

[0172] The xylanase used in this assay is Bacillus subtilis wild typexylanase.

[0173] 250 μl xylanase solution containing 12 TXU/ml, approx. 100 μlxylanase inhibitor solution and citric acid (0.1 M)-di-sodium-hydrogenphosphate (0.2 M) buffer, pH 5, to react a reaction volume of 1000 μl ispre-incubated for 5 minutes at 40° C. At t=5 minutes, 1 Xylazyme(Megazyme, Ireland) tablet is added to the reaction mixture. At t=15minutes the reaction is terminated, by addition of 10 ml 2% TRIS/NaOH,pH 12. The solution is filtered and the absorbency of the supernatant ismeasured at 590 nm. By choosing several different concentrations ofinhibitor in the above assay, it is possible to create a plot of ODversus inhibitor concentration. Using the slope (a) and intercept (b)from this plot and the concentration of the xylanase it is possible tocalculate the amount of XIU in a given inhibitor solution (equation 1).

amount of XIU in solution=((b/2)/−a)/TXU  Equation 1

[0174] From the endogenous xylanase inhibitor purification, thefollowing inhibitor yield was recovered (table 1). TABLE 1 Wheatendogenous xylanase inhibitor recovery after purification. Sample AmountXIU XIU, total Recovery, % Flour 2000 g 590/g 1.180.000 100 Purifiedinhibitor 90 ml 4658/ml 419.220  35.5

[0175] The inhibitor sample was pure and free from wheat endogenousxylanolytic activities.

Example 2 Fractionation and Reconstruction of Wheat Flour Free ofXylanase Inhibitor and Xylanases Functionality in this Flour as aFunction of Added Xylanase Inhibitor

[0176] Flour Fractionation and Reconstitution

[0177] The flour used was: Danish Reform flour, batch No 99056. Thefractionation, inhibitor inactivation and reconstitution were asfollows:

[0178] A simple dough was made by mixing 1600 gram flour, with optimalwater addition, according to a baker's absorption at 500 BU and mixingtime according to Farinograph results. This resulted in 2512 gram dough.The gluten was manually washed out from the dough, using a water doughratio of approx. 5:1. The water used was pre-chilled to 4° C. to preventfurther enzyme activity in the dough. The resulting wash-water containedthe soluble proteins (including the xylanase inhibitor), lipids,non-starch polysaccharides and starch. The starch and other non-solublecomponents were separated from the wash-water by centrifugation (5000 g,10 minutes, 10° C.). To inactivate the endogenous xylanase inhibitor inthe wash-water, the supernatant from the centrifugation was boiled forthree minutes using a heat-evaporator.

[0179] All three fractions (gluten, starch and solubles) were frozen inflasks and placed in a freeze dryer. After drying, the fractions wereweighed, grounded using a mortar and pestle, coffee mill and sievedthrough a 250 μm sieve. All fractions were weighed again and flour wasreconstituted, based on the ratios obtained after fractionation.

[0180] Enzymes

[0181] The xylanases listed in table 2 have been used in the study. Thexylanases are purified, meaning no other xylonolytic activity is presentin the sample. TABLE 2 Xylanases used in the study and activity, TXU. IDOrigin TXU B. sub B. subtilis. 5100 A. nig A. niger 8800

[0182] Xylanase Assay (Endo-β-1,4-Xylanase Activity)

[0183] Xylanase samples are diluted in citric acid (0.1M)-di-sodium-hydrogen phosphate (0.2 M) buffer, pH 5.0, to obtainapprox. OD=0.7 in the final assay. Three dilutions of the sample and aninternal standard with a defined activity are thermostated for 5 minutesat 40° C. At time=5 minutes, 1 Xylazyme tab (crosslinked, dyed xylansubstrate) is added to the enzyme solution. At time=15 minutes (or insome cases longer, depending on the xylanase activity present in thesample) the reaction is terminated, by adding 10 ml of 2% TRIS. Thereaction mixture is centrifuged and the OD of the supernatant ismeasured at 590 nm. Taking into account the dilutions and the amount ofxylanase, the activity (TXU, Total-Xylanase-Units) of the sample can becalculated relative to the standard.

[0184] Baking Trials

[0185] Baking trials were done with (1.44×initial inhibitor level inDanish Reform flour, batch No 99056) and without addition of purifiedendogenous xylanase inhibitor to the reconstituted flour, respectively.The baking trials were done using the xylanases listed in table 2 andthe compositions listed in table 3.. TABLE 3 Composition of dough madewithin the baking trials. Dough No ID TXU Inh. add, XIU/50 g 1 Control 00 2 B. sub 7500 0 3 A. nig 7500 0 4 B. sub 7500 850 5 A. nig 7500 850 6Control 0 850

[0186] Dough Analysis

[0187] The dough were analysed with respect to:

[0188] Stickiness

[0189] Dough stickiness was measured on a TX-XT2 system (Stable MicroSystems) using a SMS Dough Stickiness Cell according to the methoddescribed by Chen And Hoseney (Lebensmittel Wiss u.-Technol., 28,467-473. 1995).

[0190] Viscosity Analysis of Dough Liquid

[0191] The viscosity of extracted dough liquid was measured using aBrookfield viscosimeter after extraction.

[0192] Pentosan Analysis of Dough Liquid

[0193] Solubilised pentosan was measured in the dough liquid using themethod of Rouau and Surget (Carbohydrate polymers, 24, 123-132, 1994).

[0194] Results

[0195] Flour Fractionation and Reconstitution

[0196] The fractionation and reconstitution of the dough resulted in168.15 grams of freeze dried gluten, 111.13 grams of freeze driedsoluble fraction and 1143.56 grams of freeze dried starch.

[0197] Inhibitor Quantification in Flour

[0198] Using the inhibitor quantification method, the inhibitor level inthe 99056 flour and the reconstituted flour could be detected. Theresults from these analyses are listed in table 4. TABLE 4 Results frominhibitor quantification in native flour (99056) and reconstitutedflour. Flour Inhibitor concentration, XIU/g flour 99056 590Reconstituted flour 42

[0199] Comparing the inhibitor level in the two portions of flour a 93%(100-(42XIU/590XIU)×100%)) decrease of inhibitor level in thereconstituted flour is shown.

[0200] Baking Trials

[0201] The results from the baking trial are listed in tables 5 and 6.TABLE 5 Data from baking trials with reconstituted flour, xylanase and+/− xylanase inhibitor addition. Std. dev., % represents the standarddeviation over two days of baking. Avg. spec. vol, ID TXU Inh., XIU/50 gml/gram Std. dev., % Control 0 42 3.04 4.06 B. sub 7500 42 3.23 12.51 A.nig. 7500 42 3.44 5.24 B. sub 7500 850 3.22 4.26 A. nig. 7500 850 3.380.70 Control 0 850 2.94 0.05

[0202] The standard deviation shown in table 5 reflects the doughhandling properties of the tested dough. The dough made without theendogenous xylanase inhibitor (42 XIU), were very difficult to handle.The standard deviation for these doughs are in the area of 3 to 12.5%.Compared to the dough with the inhibitor added, this is quite high. Ifthese standard deviations are compared with the actual changes in thebread volume, it can be seen that the figures are approximately the samevalue. This means that we can not conclude anything about the absence ofthe inhibitor's influence on the bread volume. If we look at the doughmade with addition of the endogenous xylanase inhibitor (850 XIU) intable 5, we can see that we were able to produce bread from thereconstituted flour in a reproducible way over a two day period. Thestandard deviation was within the area of 0.05 to 4.2%, which isacceptable. From table 6 it can be seen, that the xylanases allincreased the volume of the baked bread. TABLE 6 Volume increase inbread baked from reconstituted flour as a function of xylanase andxylanase inhibitor addition. Inh., Avg. spec. vol, Volume increase as IDTXU XIU/50 g ml/gram function of xylanase, % Control 0 42 3.04 0.0 B.sub 7500 42 3.23 6.2 A. nig. 7500 42 3.44 13.3 B. sub 7500 850 3.22 9.7A. nig. 7500 850 3.38 15.0 Control 0 850 2.94 0.0

[0203] What can be deduced from table 5 and table 6, is that the absenceof the xylanase inhibitor in the flour made the handling of the doughvery difficult. Therefore, what may seem as a positive response involume by addition of inhibitor in table 6, probably can be explained bythe high standard deviation in the dough lacking the inhibitor, due todifficult handling properties. Furthermore, it can be concluded that allthe xylanases tested increased the bread volume significantly comparedto the blank control.

[0204] Stickiness

[0205] The same dough, that was used for the baking trials, was used forstickiness measurements. The results are listed in table 7. TABLE 7 Datarepresenting stickiness as a function of time, xylanase and xylanaseinhibitor addition to reconstituted flour. Inh., Avg. stickiness afterAvg. stickiness ID TXU XIU/50 g 10 min, g × s after 60 min, g × sControl 0 42 4.71 4.79 B. sub. 7500 42 12.20 13.39 A. nig. 7500 42 9.2212.58 B. sub. 7500 850 2.51 3.66 A. nig. 7500 850 5.24 6.45 Control 0850 4.10 4.15

[0206] The results in table 7 clearly indicate the influence of theinhibitor that was observed in the experiment. The dough with a lowlevel of xylanase inhibitor in combination with xylanase, was verydifficult to handle and mould. However, when the inhibitor was added,the dough became dry and very easy to handle. As can be seen from table7, addition of the 990202 xylanase in combination with the inhibitordecreased the stickiness. The dough became drier.

[0207] Table 7 also shows that there is only a small effect of time onthe stickiness. It seems that the xylanases act very rapidly. Within thefirst 10 minutes most of the arabinoxylan is modified when the firstxylanase (B. sub) is added. The second xylanase tested (A. nig), seemsto act less rapidly. A function of time can easily be observed usingthis xylanase. This is also the xylanase that shows the least effect asa function of inhibitor level when analysed on stickiness.

[0208] Dough Viscosity

[0209] The dough viscosity and the pentosan analysis results wereobtained from the same extraction of dough prepared from reconstitutedflour added xylanase and xylanase inhibitor. This dough was analysedafter two proofing times, 30 and 120 minutes.

[0210] The results of the viscosity analysis are presented in table 8.TABLE 8 Data representing dough liquid viscosity as a function of time,xylanase and xylanase inhibitor addition to reconstituted flour. Avg.dough Inh., viscosity, cP, Avg. dough viscosity, ID TXU XIU/50 g 30 minproofing cP, 120 min proofing Control 0 42 5.21 5.56 B. sub. 7500 425.07 4.55 A. nig. 7500 42 5.78 4.14 B. sub. 7500 850 9.03 11.09 A. nig.7500 850 8.44 8.55 Control 0 850 5.96 6.95

[0211] As can be seen from table 8 the inhibitor has a significanteffect on the functionality of the xylanases. Without addition of theinhibitor, the arabinoxylan is being de-polymerised to Low MolecularWeight (LMW) arabinoxylan with a low viscosity. Addition of inhibitorprevents this very extensive de-polymerisation of the arabinoxylan.

[0212] Pentosan Analysis of Dough Liquid

[0213] The results from the pentosan (arabinoxylan) analysis of thedough liquid are presented in table 9. TABLE 9 Data representingpentosan solubilisation as a function of time, xylanase and xylanaseinhibitor addition to reconstituted flour. Inh., Avg. Pentosan, %, Avg.Pentosan, %, ID TXU XIU/50 g 30 min proofing 120 min proofing Control 042 0.387 0.458 B. sub. 7500 42 0.766 0.819 A. nig. 7500 42 0.719 0.798B. sub. 7500 850 0.410 0.544 A. nig. 7500 850 0.560 0.673 Control 0 8500.400 0.528

[0214] As can be seen from the results in table 9, the addition ofendogenous xylanase inhibitor decreased the solubilisation of thearabinoxylan. When evaluated after 30 minutes proofing time, the amountof arabinoxylan solubilised in absence of the inhibitor is almost twicethe amount as in presence of the inhibitor. Calculated on the basis ofthe relating control samples, the solubilisation is much higher inabsence of the inhibitor, as illustrated in the following example:

(0.766−0.387)/(0.410−0.400)=37.9 times higher solubilisation

[0215] The above example was calculated on basis of solubilisation ofarabinoxylan using the Bacillus xylanase, 30 minutes proofing and+/−inhibitor.

Example 3 Site-Directed Mutagenesis on Xylanases

[0216] Specific mutants of Bacillus subtilis xylanase may be obtained bysite directed mutagenesis of the wild type enzyme, by the use of any ofa number of commercially available mutagenesis kits. An example of howto obtain the D11F mutant using the Quick Exchange kit, available fromStratagene Cloning Systems, 11011 North Torrey Pines Road, La Jolla,Calif. 92037, USA is given below:

[0217] The DNA sequence encoding Bacillus subtilis xylanase A has beenpublished by Paice et al., 1986.

[0218] The sequence of the coding region is as follows, with thesequence encoding the mature part of the protein shown in capitals:catatgtttaagtttaaaaagaatttcttagttggattatcggcagctttaatgagtattagcttgttttcggcaaccgcctctgcaGCTAGCACAGACTACTGGCAAAATTGGACTGATGGGGGCGGTATAGTAAACGCTGTCAATGGGTCTGGCGGGAATTACAGTGTTAATTGGTCTAATACCGGAAATTTTGTTGTTGGTAAAGGTTGGACTACAGGTTCGCCATTTAGGACGATAAACTATAATGCCGGAGTTTGGGCGCCGAATGGCAATGGATATTTAACTTTATATGGTTGGACGAGATCACCTCTCATAGAATATTATGTAGTGGATTCATGGGGTACTTATAGACCTACTGGAACGTATAAAGGTACTGTAAAAAGTGATGGGGGTACATATGACATATATACAACTACACGTTATAACGCACCTTCCATTGATGGCGATCGCACTACTTTTACGCAGTACTGGAGTGTTCGCCAGTCGAAGAGACCAACCGGAAGCAACGCTACAATCACTTTCAGCAATCATGTGAACGCATGGAAGAGCCATGGAATGAATCTGGGCAGTAATTGGGCTTACCAAGTCATGGCGACAGAAGGATATCAAAGTAGTGGAAGTTCTAACGTAACAGTGTGGTAA

[0219] The part of the gene encoding the mature part of the wild typeenzyme may be expressed intracellularly in E.coli by methods well knownto people skilled in the art of molecular biology. For example:

[0220] 1. Generating a copy of the capitalised part of the abovedescribed gene by use of the Polymerase Chain Reaction (PCR) with anadded NdeI restriction enzyme site (CATATG) before the GCTAGCACA and anadded HindIII restriction site (AAGCTT) after the GTGTGGTAA.

[0221] 2. Inserting the resultant modified copy of the gene by use ofthe above mentioned enzymes into the expression vector pET24a(+), whichcan be obtained from Novagen, Inc. 601 Science Drive, Madison, Wis.53711, USA.

[0222] 3. Transforming into a suitable E.coli strain and expression byfermentation as described by the vendor of pET24a(+).

[0223] Our D11F mutant enzyme may be obtained by using the “QuickExchange” mutagenesis kit according to the manufacturer, and using theabove described Bacillus subtilis wild type xylanase-pET24a(+) constructand the following PCR mutagenesis primers:

[0224] Sense primer:

[0225] CTACTGGCAAAATTGGACTTTTGGAGGAGGTATAGTAAACGCTG

[0226] Antisense primer:

[0227] CAGCGTTTACTATACCTCCTCCAAAAGTCCAATTTTGCCAGTAG

[0228] The mutant enzyme is expressed and purified using the sameprotocols as for the wild type enzyme.

Example 4 Inhibition Studies of Xylanase Mutants

[0229] Xylanase mutants expressed in E. coli (see Example 3) werefermented and purified (meaning no other xylanolytic activity waspresent in the purified preparation) using a de-salting step and acation exchange chromatography step.

[0230] These pure xylanase mutant preparations were diluted to 12 TXU/mlusing 0.1 M citric acid-0.2 M di-sodium-hydrogen phosphate, pH 5.0 andused in the following assay.

[0231] A stable inhibitor preparation was made according to the protocoldescribed in Example 1. This stable inhibitor preparation is used asstock for all xylanase-xylanase inhibitor studies. Using the inhibitorquantification method described in example 1, the inhibitor preparationwas analysed to contain 126 XIU/ml.

[0232] Assay

[0233] To 250 μl diluted xylanase mutant preparations, are added 0, 10,25, 50 or 100 μl inhibitor preparation, respectively. To theseinhibitor-xylanase mixtures were added 0.1 M citric acid-0.2 Mdi-sodium-hydrogen phosphate, pH 5.0 making the end-volume 1000 μl.These reaction mixtures were pre-incubated for 5 minutes at 40° C.Hereafter 1 xylazyme tablet (Megazyme, Ireland) were added to allinhibitor-xylanase mixtures. After 10 minutes of incubation at 40° C.,the reactions were terminated, by adding 10 ml 2% Tris/NaOH, pH 12.0.The mixtures were centrifuged and the liberated blue colour from thesubstrate was measured at 590 nm.

[0234] The results are presented in table 10. TABLE 10 Relativeinhibition of xylanase mutants and parent xylanase (here wildtypeenzyme) as a function of xylanase inhibitor. Mutant ID 0 1, 26 3, 15 6,3 12, 6 Relative inhibition, % Wildtype 100 77 48 29 23 D11Y 100 120 114126 124 D11N 100 93 72 53 32 D11F 100 114 119 116 115 D11K 100 109 112113 116 D11S 100 98 81 60 38 D11W 100 101 88 70 50 G34D 100 94 83 70 53G34F 100 76 53 34 29 G34T 100 99 99 93 86 Y113A 100 96 80 62 43 Y113D100 96 81 63 45 Y113K 100 103 85 63 47 N114A 100 80 49 28 22 N114D 10084 57 39 29 N114F 100 84 54 39 34 N114K 100 87 56 33 24 D121N 100 80 3616 14 D121K 100 104 95 85 75 D121F 100 101 89 72 60 D121A 100 81 50 2721 R122D 100 85 59 41 28 R122F 100 93 74 58 58 R122A 100 78 46 33 26Q175E 100 87 59 40 31 Q175S 100 88 59 30 19 Q175L 100 78 42 25 23 G12F100 110 106 100 92 G13F 100 104 95 87 84 I15K 100 84 47 28 23 N32K 10082 42 19 14 G120K 100 85 52 29 22 G120D 100 84 47 24 18 G120F 100 71 3518 15 G120Y 100 81 40 18 16 G120N 100 84 49 29 23 D119K 100 94 67 40 26D119Y 100 87 50 28 22 D119N 100 91 74 44 22 T123K 100 80 46 30 25 T123Y100 80 47 28 27 T123D 100 83 36 20 17 T124K 100 110 92 73 57 T124Y 100101 76 49 33 T124D 100 87 52 32 25 N17K 100 88 48 31 26 N17Y 100 79 4223 19 N17D 100 90 81 50 22 N29K 100 83 50 30 23 N29Y 100 85 49 30 24N29D 100 74 44 26 20 S31K 100 77 42 23 23 S31Y 100 83 50 27 22 S31D 10079 52 30 24 D11F/R122D 100 109 111 110 109 D11F/G34D 100 104 106 103 104

[0235] From the results in table 10, it can be seen the xylanase mutantsD11Y, D11F, D11K, D11F/R122D and D11F/G34D are uninhibited by the wheatendogenous xylanase inhibitor. These xylanase mutants would be expectedto act more aggressively/specifically on the soluble arabinoxylan,compared to the other xylanase mutants or other xylanases. They wouldtherefore be superior in applications where a decrease in viscosity (asa function of HMW arabinoxylan) is wanted.

Example 5 Functionality Studies of Xylanase Mutants

[0236] Xylanase mutants expressed in E. coli (see Example 3) werefermented and purified (meaning no other xylanolytic activity werepresent in the purified preparation).

[0237] These pure xylanase mutant preparations were diluted to 400TXU/ml using water and used in the following assay.

[0238] Assay

[0239] 200 ml 30% (w/w) flour slurry was made using water (thermostatedto 25° C.), by stirring for 5 minutes. 60.0 ml of this flour slurry ispoured into a Ford-cup, and the time for drainage of 50.0 ml ismeasured. This measurement is the blank measurement. The 60.0 ml flourslurry is poured back and 1000 μl diluted xylanase mutant preparation isadded to the flour slurry under stirring. After 2, 5, 10 and 20 minutes,60.0 ml is poured into the Ford-cup, and the drainage time for 50.0 mlis recorded. Each measurement were done in triplicate.

[0240] The results are presented in table 11. TABLE 11 Relativeviscosity of flour slurry as a function of xylanase mutant and parentxylanase (here wild type xylanase) Incubation time, minutes Mutant ID 02 5 10 20 Relative viscosity change, % Wildtype 100 112 120 131 141 D11Y100 97 93 83 75 D11N 100 112 125 130 136 D11F 100 93 87 78 69 D11K 100105 95 88 78 D11S 100 102 110 113 117 D11W 100 106 115 121 122 G34D 100110 120 128 124 G34F 100 111 126 128 146 G34T 100 100 108 111 106 Y113A100 118 129 130 124 Y113D 100 116 127 124 114 Y113K 100 118 123 121 115N114A 100 117 128 127 131 N114D 100 125 144 162 170 N114F 100 113 119131 150 N114K 100 119 129 141 147 D121N 100 104 103 106 104 D121K 100122 132 141 162 D121F 100 107 117 128 147 D121A 100 101 102 103 107R122D 100 120 119 124 115 R122F 100 127 144 150 160 R122A 100 123 138144 153 Q175E 100 116 134 142 149 Q175S 100 110 113 121 129 Q175L 100111 111 119 126 G12F 100 127 132 122 101 G13F 100 106 119 124 113 I15K100 109 108 113 118 N32K 100 97 98 101 101 G120K 100 103 111 115 121G120D 100 112 122 120 126 G120F 100 103 111 117 130 G120Y 100 106 106108 126 G120N 100 119 123 130 141 D119K 100 118 119 127 125 D119Y 100102 102 111 110 D119N 100 126 137 145 146 T123K 100 106 109 121 120T123Y 100 101 106 108 116 T123D 100 113 123 125 126 T124K 100 117 131128 127 T124Y 100 112 123 132 135 T124D 100 103 110 111 118 N17K 100 114119 119 132 N17Y 100 102 102 108 108 N17D 100 120 131 135 143 N29K 10098 100 100 104 N29Y 100 115 117 132 143 N29D 100 104 104 113 111 S31K100 119 115 124 134 S31Y 100 110 118 122 137 S31D 100 99 103 109 110D11F/R122D 100 91 89 82 77 D11F/G34D 100 96 93 84 80

Example 6 Site-Directed Mutation in the Active Site of Bacillus subtilisXylanase A, Does Not Influence the Xylanase: Xylanase InhibitorInteraction

[0241] A residue in the active site of the Bacillus subtilis wildtypexylanase A enzyme was altered by a site-directed mutation (see ex. 3) Inthe mutated residue (Y166F) a potential hydrogen bond is lost. Themutant xylanase, was expressed in E coli, fermented and purified.Hereafter, the mutant was investigated for its interaction with thexylanase inhibitor (see example 4).

[0242] As can be seen below (table 12), the exchange of an amino acid inthe active site, did surprisingly not have any effect on interactionswith the xylanase inhibitor as compared to the Bacillus subtiliswildtype xylanase enzyme. TABLE 12 Relative inhibition of Bacillussubtilis wildtype xylanase and the xylanase mutant Y166F. XIU/mlXylanase ID 0 1, 26 3, 15 6, 3 12, 6 Relative inhibition, % Wildtype 10075 40 24 20 Y166F 100 74 39 22 20

[0243] Hence, in summary the experiment described above shows asite-directed mutation in the active site of the Bacillus subtilisxylanase A, which mutation does not influence the xylanase'sinteractions with the xylanase inhibitor.

Example 7 Site-Directed Mutation in Family 11 Xylanases Other Than theBacillus subtilis Xylanase A, Influencing the Xylanase-XylanaseInhibitor Interactions

[0244] D19 residue of the Thermomyces lanuginosus xylanase A enzyme wasmutated to F19 by site-directed mutagenesis. D19 corresponds to D11residue in the Bacillus subtilis xylanase (SEQ ID NO. 1). Thermomyceslanuginosus xylanase A gene is described as SEQ ID NO. 9.

[0245] The primers for PCR construction of the D19F mutant may be thefollowing:

[0246] Sense primer:

[0247] GGTTATTACTATTCCTGGTGGAGTTTTGGAGGAGCGCAGGCCACG

[0248] Antisense primer:

[0249] CGTGGCCTGCGCTCCTCCAAAACTCCACCAGGAATAGTAATAACC

[0250] The obtained mutant xylanase (D19F), was expressed in E. coli,fermented and purified. Hereafter, the mutant and the Thermomyceslanuginosus wildtype xylanase A was investigated for to its interactionwith the xylanase inhibitor (see example 4). As can be seen from theresults in table 13, the D19F mutant of the Thermomyces lanuginosusxylanase A is significantly less inhibited by the xylanase inhibitor ascompared to the Thermomyces lanuginosus wildtype xylanase A. TABLE 13Relative inhibition of Thermomyces lanoginosus wildtype xylanase A (TLX)and the Thermomyces lanoginosus mutant xylanase, D19F (D19F). XIU/mlXylanase ID 0 1, 26 3, 15 6, 3 12, 6 Relative inhibition, % TLX 100 4524 17 14 D19F 100 73 38 24 20

[0251] Hence, in summary the experiment described above shows asite-directed mutation in the Thermomyces lanuginosus xylanase A. Theresults show that a mutation introducing a substitution of an amino acidon the surface of the xylanase molecule (analogue to the D11F in B.subtilis) changes the xylanase:xylanase inhibitor interactions. Thus,our invention (i.e. that surface residues control the level ofinhibition of xylanase) holds true for xylanases that are homologous tothe B. subtilis xylanase.

SUMMARY

[0252] In summary, the present invention provides a means for alteringthe sensitivity of a xylanase enzyme to a xylanase inhibitor.

[0253] All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the present invention. Although the present invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biochemistry and biotechnology or related fields areintended to be within the scope of the following claims.

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[0267] Slade, L., Levine, H., Craig, S., Arciszewski, H. and Saunders,S. (1993). Enzyme treated low moisture content comestible products. U.S.Pat. No. 5,200,215 by Nabisco.

[0268] Soerensen, J. F. and Sibbesen, O. (1999). Bacterial xylanase. UKA 9828599.2.

1 66 1 185 PRT Bacillus subtilis 1 Ala Ser Thr Asp Tyr Trp Gln Asn TrpThr Asp Gly Gly Gly Ile Val 1 5 10 15 Asn Ala Val Asn Gly Ser Gly GlyAsn Tyr Ser Val Asn Trp Ser Asn 20 25 30 Thr Gly Asn Phe Val Val Gly LysGly Trp Thr Thr Gly Ser Pro Phe 35 40 45 Arg Thr Ile Asn Tyr Asn Ala GlyVal Trp Ala Pro Asn Gly Asn Gly 50 55 60 Tyr Leu Thr Leu Tyr Gly Trp ThrArg Ser Pro Leu Ile Glu Tyr Tyr 65 70 75 80 Val Val Asp Ser Trp Gly ThrTyr Arg Pro Thr Gly Thr Tyr Lys Gly 85 90 95 Thr Val Lys Ser Asp Gly GlyThr Tyr Asp Ile Tyr Thr Thr Thr Arg 100 105 110 Tyr Asn Ala Pro Ser IleAsp Gly Asp Arg Thr Thr Phe Thr Gln Tyr 115 120 125 Trp Ser Val Arg GlnSer Lys Arg Pro Thr Gly Ser Asn Ala Thr Ile 130 135 140 Thr Phe Ser AsnHis Val Asn Ala Trp Lys Ser His Gly Met Asn Leu 145 150 155 160 Gly SerAsn Trp Ala Tyr Gln Val Met Ala Thr Glu Gly Tyr Gln Ser 165 170 175 SerGly Ser Ser Asn Val Thr Val Trp 180 185 2 35 PRT wheat 2 Gly Ala Pro ValAla Arg Ala Val Glu Ala Val Ala Pro Phe Gly Val 1 5 10 15 Cys Tyr AspThr Lys Thr Leu Gly Asn Asn Leu Gly Gly Tyr Ala Val 20 25 30 Pro Asn Val35 3 17 PRT Wheat 3 Lys Arg Leu Gly Phe Ser Arg Leu Pro His Phe Thr GlyCys Gly Gly 1 5 10 15 Leu 4 21 PRT Wheat 4 Leu Pro Val Pro Ala Pro ValThr Lys Asp Pro Ala Thr Ser Leu Tyr 1 5 10 15 Thr Ile Pro Phe His 20 531 PRT Wheat 5 Leu Leu Ala Ser Leu Pro Arg Gly Ser Thr Gly Val Ala GlyLeu Ala 1 5 10 15 Asn Ser Gly Leu Ala Leu Pro Ala Gln Val Ala Ser AlaGln Lys 20 25 30 6 24 PRT Wheat 6 Gly Gly Ser Pro Ala His Tyr Ile SerAla Arg Phe Ile Glu Val Gly 1 5 10 15 Asp Thr Arg Val Pro Ser Val Glu 207 13 PRT Wheat 7 Val Asn Val Gly Val Leu Ala Ala Cys Ala Pro Ser Lys 1 510 8 41 PRT Wheat 8 Val Ala Asn Arg Phe Leu Leu Cys Leu Pro Thr Gly GlyPro Gly Val 1 5 10 15 Ala Ile Phe Gly Gly Gly Pro Val Pro Trp Pro GlnPhe Thr Gln Ser 20 25 30 Met Pro Tyr Thr Leu Val Val Val Lys 35 40 9 588DNA Thermomyces lanuginosus 9 atgcagacaa cccccaactc ggagggctggcacgatggtt attactattc ctggtggagt 60 gacggtggag cgcaggccac gtacaccaacctggaaggcg gcacctacga gatcagctgg 120 ggagatggcg gtaacctcgt cggtggaaagggctggaacc ccggcctgaa cgcaagagcc 180 atccactttg agggtgttta ccagccaaacggcaacagct accttgcggt ctacggttgg 240 acccgcaacc cgctggtcga gtattacatcgtcgagaact ttggcaccta tgatccttcc 300 tccggtgcta ccgatctagg aactgtcgagtgcgacggta gcatctatcg actcggcaag 360 accactcgcg tcaacgcacc tagcatcgacggcacccaaa ccttcgacca atactggtcg 420 gtccgccagg acaagcgcac cagcggtaccgtccagacgg gctgccactt cgacgcctgg 480 gctcgcgctg gtttgaatgt caacggtgaccactactacc agatcgttgc aacggagggc 540 tacttcagca gcggctatgc tcgcatcaccgttgctgacg tgggctaa 588 10 645 DNA Bacillus subtilis 10 catatgtttaagtttaaaaa gaatttctta gttggattat cggcagcttt aatgagtatt 60 agcttgttttcggcaaccgc ctctgcagct agcacagact actggcaaaa ttggactgat 120 gggggcggtatagtaaacgc tgtcaatggg tctggcggga attacagtgt taattggtct 180 aataccggaaattttgttgt tggtaaaggt tggactacag gttcgccatt taggacgata 240 aactataatgccggagtttg ggcgccgaat ggcaatggat atttaacttt atatggttgg 300 acgagatcacctctcataga atattatgta gtggattcat ggggtactta tagacctact 360 ggaacgtataaaggtactgt aaaaagtgat gggggtacat atgacatata tacaactaca 420 cgttataacgcaccttccat tgatggcgat cgcactactt ttacgcagta ctggagtgtt 480 cgccagtcgaagagaccaac cggaagcaac gctacaatca ctttcagcaa tcatgtgaac 540 gcatggaagagccatggaat gaatctgggc agtaattggg cttaccaagt catggcgaca 600 gaaggatatcaaagtagtgg aagttctaac gtaacagtgt ggtaa 645 11 657 DNA Unknown B.subtilis xylanase sequence with added restriction site 11 catatgtttaagtttaaaaa gaatttctta gttggattat cggcagcttt aatgagtatt 60 agcttgttttcggcaaccgc ctctgcacat atggctagca cagactactg gcaaaattgg 120 actgatgggggcggtatagt aaacgctgtc aatgggtctg gcgggaatta cagtgttaat 180 tggtctaataccggaaattt tgttgttggt aaaggttgga ctacaggttc gccatttagg 240 acgataaactataatgccgg agtttgggcg ccgaatggca atggatattt aactttatat 300 ggttggacgagatcacctct catagaatat tatgtagtgg attcatgggg tacttataga 360 cctactggaacgtataaagg tactgtaaaa agtgatgggg gtacatatga catatataca 420 actacacgttataacgcacc ttccattgat ggcgatcgca ctacttttac gcagtactgg 480 agtgttcgccagtcgaagag accaaccgga agcaacgcta caatcacttt cagcaatcat 540 gtgaacgcatggaagagcca tggaatgaat ctgggcagta attgggctta ccaagtcatg 600 gcgacagaaggatatcaaag tagtggaagt tctaacgtaa cagtgtggta aaagctt 657 12 44 DNAUnknown sense primer 12 ctactggcaa aattggactt ttggaggagg tatagtaaac gctg44 13 44 DNA Unknown Antisense primer 13 cagcgtttac tatacctcctccaaaagtcc aattttgcca gtag 44 14 45 DNA Unknown Sense primer 14ggttattact attcctggtg gagttttgga ggagcgcagg ccacg 45 15 45 DNA UnknownAntisense primer 15 cgtggcctgc gctcctccaa aactccacca ggaatagtaa taacc 4516 213 PRT Bacillus subtilis 16 Met Phe Lys Phe Lys Lys Asn Phe Leu ValGly Leu Ser Ala Ala Leu 1 5 10 15 Met Ser Ile Ser Leu Phe Ser Ala ThrAla Ser Ala Ala Ser Thr Asp 20 25 30 Tyr Trp Gln Asn Trp Thr Asp Gly GlyGly Ile Val Asn Ala Val Asn 35 40 45 Gly Ser Gly Gly Asn Tyr Ser Val AsnTrp Ser Asn Thr Gly Asn Phe 50 55 60 Val Val Gly Lys Gly Trp Thr Thr GlySer Pro Phe Arg Thr Ile Asn 65 70 75 80 Tyr Asn Ala Gly Val Trp Ala ProAsn Gly Asn Gly Tyr Leu Thr Leu 85 90 95 Tyr Gly Trp Thr Arg Ser Pro LeuIle Glu Tyr Tyr Val Val Asp Ser 100 105 110 Trp Gly Thr Tyr Arg Pro ThrGly Thr Tyr Lys Gly Thr Val Lys Ser 115 120 125 Asp Gly Gly Thr Tyr AspIle Tyr Thr Thr Thr Arg Tyr Asn Ala Pro 130 135 140 Ser Ile Asp Gly AspArg Thr Thr Phe Thr Gln Tyr Trp Ser Val Arg 145 150 155 160 Gln Ser LysArg Pro Thr Gly Ser Asn Ala Thr Ile Thr Phe Ser Asn 165 170 175 His ValAsn Ala Trp Lys Ser His Gly Met Asn Leu Gly Ser Asn Trp 180 185 190 AlaTyr Gln Val Met Ala Thr Glu Gly Tyr Gln Ser Ser Gly Ser Ser 195 200 205Asn Val Thr Val Trp 210 17 213 PRT Bacillus circulans 17 Met Phe Lys PheLys Lys Asn Phe Leu Val Gly Leu Ser Ala Ala Leu 1 5 10 15 Met Ser IleSer Leu Phe Ser Ala Thr Ala Ser Ala Ala Ser Thr Asp 20 25 30 Tyr Trp GlnAsn Trp Thr Asp Gly Gly Gly Ile Val Asn Ala Val Asn 35 40 45 Gly Ser GlyGly Asn Tyr Ser Val Asn Trp Ser Asn Thr Gly Asn Phe 50 55 60 Val Val GlyLys Gly Trp Thr Thr Gly Ser Pro Phe Arg Thr Ile Asn 65 70 75 80 Tyr AsnAla Gly Val Trp Ala Pro Asn Gly Asn Gly Tyr Leu Thr Leu 85 90 95 Tyr GlyTrp Thr Arg Ser Pro Leu Ile Glu Tyr Tyr Val Val Asp Ser 100 105 110 TrpGly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys Gly Thr Val Lys Ser 115 120 125Asp Gly Gly Thr Tyr Asp Ile Tyr Thr Thr Thr Arg Tyr Asn Ala Pro 130 135140 Ser Ile Asp Gly Asp Arg Thr Thr Phe Thr Gln Tyr Trp Ser Val Arg 145150 155 160 Gln Ser Lys Arg Pro Thr Gly Ser Asn Ala Thr Ile Thr Phe ThrAsn 165 170 175 His Val Asn Ala Trp Lys Ser His Gly Met Asn Leu Gly SerAsn Trp 180 185 190 Ala Tyr Gln Val Met Ala Thr Glu Gly Tyr Gln Ser SerGly Ser Ser 195 200 205 Asn Val Thr Val Trp 210 18 211 PRT Bacillusstearothermophilus 18 Met Lys Leu Lys Lys Lys Met Leu Thr Leu Leu LeuThr Ala Ser Met 1 5 10 15 Ser Phe Gly Leu Phe Gly Ala Thr Ser Ser AlaAla Thr Asp Tyr Trp 20 25 30 Gln Tyr Trp Thr Asp Gly Gly Gly Met Val AsnAla Val Asn Gly Pro 35 40 45 Gly Gly Asn Tyr Ser Val Thr Trp Gln Asn ThrGly Asn Phe Val Val 50 55 60 Gly Lys Gly Trp Thr Val Gly Ser Pro Asn ArgVal Ile Asn Tyr Asn 65 70 75 80 Ala Gly Ile Trp Glu Pro Ser Gly Asn GlyTyr Leu Thr Leu Tyr Gly 85 90 95 Trp Thr Arg Asn Ala Leu Ile Glu Tyr TyrVal Val Asp Ser Trp Gly 100 105 110 Thr Tyr Arg Ala Thr Gly Asn Tyr GluSer Gly Thr Val Asn Ser Asp 115 120 125 Gly Gly Thr Tyr Asp Ile Tyr ThrThr Met Arg Tyr Asn Ala Pro Ser 130 135 140 Ile Asp Gly Thr Gln Thr PheGln Gln Phe Trp Ser Val Arg Gln Ser 145 150 155 160 Lys Arg Pro Thr GlySer Asn Val Ser Ile Thr Phe Ser Asn His Val 165 170 175 Asn Ala Trp ArgSer Lys Gly Met Asn Leu Gly Ser Ser Trp Ala Tyr 180 185 190 Gln Val LeuAla Thr Glu Gly Tyr Gln Ser Ser Gly Arg Ser Asn Val 195 200 205 Thr ValTrp 210 19 211 PRT A. caviae 19 Met Phe Lys Phe Gly Lys Lys Leu Met ThrVal Val Leu Ala Ala Ser 1 5 10 15 Met Ser Phe Gly Val Phe Ala Ala ThrSer Ser Ala Ala Thr Asp Tyr 20 25 30 Trp Gln Asn Trp Thr Asp Gly Gly GlyThr Val Asn Ala Val Asn Gly 35 40 45 Ser Gly Gly Asn Tyr Ser Val Ser TrpGln Asn Thr Gly Asn Phe Val 50 55 60 Val Gly Lys Gly Trp Thr Tyr Gly ThrPro Asn Arg Val Val Asn Tyr 65 70 75 80 Asn Ala Gly Val Phe Ala Pro SerGly Asn Gly Tyr Leu Thr Phe Tyr 85 90 95 Gly Trp Thr Arg Asn Ala Leu IleGlu Tyr Tyr Val Val Asp Ser Trp 100 105 110 Gly Thr Tyr Arg Pro Thr GlyThr Tyr Lys Gly Thr Val Asn Ser Asp 115 120 125 Gly Gly Thr Tyr Asp IleTyr Thr Thr Met Arg Tyr Asn Ala Pro Ser 130 135 140 Ile Asp Gly Thr GlnThr Phe Pro Gln Tyr Trp Ser Val Arg Gln Ser 145 150 155 160 Lys Arg ProThr Gly Val Asn Ser Thr Ile Thr Phe Ser Asn His Val 165 170 175 Asn AlaTrp Pro Ser Lys Gly Met Tyr Leu Gly Asn Ser Trp Ser Tyr 180 185 190 GlnVal Met Ala Thr Glu Gly Tyr Gln Ser Ser Gly Asn Ala Asn Val 195 200 205Thr Val Trp 210 20 221 PRT C. carbonum 20 Met Val Ser Phe Thr Ser IleIle Thr Ala Ala Val Ala Ala Thr Gly 1 5 10 15 Ala Leu Ala Ala Pro AlaThr Asp Val Ser Leu Val Ala Arg Gln Asn 20 25 30 Thr Pro Asn Gly Glu GlyThr His Asn Gly Cys Phe Trp Ser Trp Trp 35 40 45 Ser Asp Gly Gly Ala ArgAla Thr Tyr Thr Asn Gly Ala Gly Gly Ser 50 55 60 Tyr Ser Val Ser Trp GlySer Gly Gly Asn Leu Val Gly Gly Lys Gly 65 70 75 80 Trp Asn Pro Gly ThrAla Arg Thr Ile Thr Tyr Ser Gly Thr Tyr Asn 85 90 95 Tyr Asn Gly Asn SerTyr Leu Ala Val Tyr Gly Trp Thr Arg Asn Pro 100 105 110 Leu Val Glu TyrTyr Val Val Glu Asn Phe Gly Thr Tyr Asp Pro Ser 115 120 125 Ser Gln SerGln Asn Lys Gly Thr Val Thr Ser Asp Gly Ser Ser Tyr 130 135 140 Lys IleAla Gln Ser Thr Arg Thr Asn Gln Pro Ser Ile Asp Gly Thr 145 150 155 160Arg Thr Phe Gln Gln Tyr Trp Ser Val Arg Gln Asn Lys Arg Ser Ser 165 170175 Gly Ser Val Asn Met Lys Thr His Phe Asp Ala Trp Ala Ser Lys Gly 180185 190 Met Asn Leu Gly Gln His Tyr Tyr Gln Ile Val Ala Thr Glu Gly Tyr195 200 205 Phe Ser Thr Gly Asn Ala Gln Ile Thr Val Asn Cys Pro 210 215220 21 227 PRT H. turcicum 21 Met Val Ser Phe Thr Ser Ile Ile Thr AlaAla Val Ala Ala Thr Gly 1 5 10 15 Ala Leu Ala Ala Pro Ala Thr Asp IleAla Ala Arg Ala Pro Ser Asp 20 25 30 Leu Val Ala Arg Gln Ser Thr Pro AsnGly Glu Gly Thr His Asn Gly 35 40 45 Cys Phe Tyr Ser Trp Trp Ser Asp GlyGly Ala Arg Ala Thr Tyr Thr 50 55 60 Asn Gly Ala Gly Gly Ser Tyr Ser ValSer Trp Gly Thr Gly Gly Asn 65 70 75 80 Leu Val Gly Gly Lys Gly Trp AsnPro Gly Thr Ala Arg Thr Ile Thr 85 90 95 Tyr Ser Gly Gln Tyr Asn Pro AsnGly Asn Ser Tyr Leu Ala Ile Tyr 100 105 110 Gly Trp Thr Arg Asn Pro LeuVal Glu Tyr Tyr Val Val Glu Asn Phe 115 120 125 Gly Thr Tyr Asp Pro SerSer Gln Ala Gln Asn Lys Gly Thr Val Thr 130 135 140 Ser Asp Gly Ser SerTyr Lys Ile Ala Gln Ser Thr Arg Thr Asn Gln 145 150 155 160 Pro Ser IleAsp Gly Thr Arg Thr Phe Gln Gln Tyr Trp Ser Val Arg 165 170 175 Gln AsnLys Arg Ser Ser Gly Ser Val Asn Met Lys Thr His Phe Asp 180 185 190 AlaTrp Ala Ser Lys Gly Met Asn Leu Gly Ser His Tyr Tyr Gln Ile 195 200 205Val Ala Thr Glu Gly Tyr Phe Ser Ser Gly Ser Ala Ser Ile Thr Val 210 215220 Asn Cys Pro 225 22 227 PRT A. pisi 22 Met Val Ser Phe Thr Ser IlePhe Thr Ala Ala Val Ala Ala Thr Gly 1 5 10 15 Ala Leu Ala Val Pro ValThr Asp Leu Ala Thr Arg Ser Leu Gly Ala 20 25 30 Leu Thr Ala Arg Ala GlyThr Pro Ser Ser Gln Gly Thr His Asn Gly 35 40 45 Cys Phe Tyr Ser Trp TrpThr Asp Gly Gly Ala Gln Ala Thr Tyr Thr 50 55 60 Asn Gly Ala Gly Gly SerTyr Ser Val Asn Trp Lys Thr Gly Gly Asn 65 70 75 80 Leu Val Gly Gly LysGly Trp Asn Pro Gly Ala Ala Arg Thr Ile Thr 85 90 95 Tyr Ser Gly Thr TyrSer Pro Ser Gly Asn Ser Tyr Leu Ala Val Tyr 100 105 110 Gly Trp Thr ArgAsn Pro Leu Ile Glu Tyr Tyr Val Val Glu Asn Phe 115 120 125 Gly Thr TyrAsp Pro Ser Ser Gln Ala Thr Val Lys Gly Ser Val Thr 130 135 140 Ala AspGly Ser Ser Tyr Lys Ile Ala Gln Thr Gln Arg Thr Asn Gln 145 150 155 160Pro Ser Ile Asp Gly Thr Gln Thr Phe Gln Gln Tyr Trp Ser Val Arg 165 170175 Gln Asn Lys Arg Ser Ser Gly Ser Val Asn Met Lys Thr His Phe Asp 180185 190 Ala Trp Ala Ala Lys Gly Met Lys Leu Gly Thr His Asn Tyr Gln Ile195 200 205 Val Ala Thr Glu Gly Tyr Phe Ser Ser Gly Ser Ala Gln Ile ThrVal 210 215 220 Asn Cys Ala 225 23 201 PRT S. commune 23 Ala Ala Ser GlyThr Pro Ser Ser Thr Gly Thr Asp Gly Gly Tyr Tyr 1 5 10 15 Tyr Ser TrpTrp Thr Asp Gly Ala Gly Asp Ala Thr Tyr Gln Asn Asn 20 25 30 Gly Gly GlySer Tyr Thr Leu Thr Trp Ser Gly Asn Asn Gly Asn Leu 35 40 45 Val Gly GlyLys Gly Trp Asn Pro Gly Ala Ala Ser Arg Ser Ile Ser 50 55 60 Tyr Ser GlyThr Tyr Gln Pro Asn Gly Asn Ser Tyr Leu Ser Val Tyr 65 70 75 80 Gly TrpThr Arg Ser Ser Leu Ile Glu Tyr Tyr Ile Val Glu Ser Tyr 85 90 95 Gly SerTyr Asp Pro Ser Ser Ala Ala Ser His Lys Gly Ser Val Thr 100 105 110 CysAsn Gly Ala Thr Tyr Asp Ile Leu Ser Thr Trp Arg Tyr Asn Ala 115 120 125Pro Ser Ile Asp Gly Thr Gln Thr Phe Glu Gln Phe Trp Ser Val Arg 130 135140 Asn Pro Lys Lys Ala Pro Gly Gly Ser Ile Ser Gly Thr Val Asp Val 145150 155 160 Gln Cys His Phe Asp Ala Trp Lys Gly Leu Gly Met Asn Leu GlySer 165 170 175 Glu His Asn Tyr Gln Ile Val Ala Thr Glu Gly Tyr Gln SerSer Gly 180 185 190 Thr Ala Thr Ile Thr Val Thr Ala Ser 195 200 24 225PRT T. lanuginosus 24 Met Val Gly Phe Thr Pro Val Ala Leu Ala Ala LeuAla Ala Thr Gly 1 5 10 15 Ala Leu Ala Phe Pro Ala Gly Asn Ala Thr GluLeu Glu Lys Arg Gln 20 25 30 Thr Thr Pro Asn Ser Glu Gly Trp His Asp GlyTyr Tyr Tyr Ser Trp 35 40 45 Trp Ser Asp Gly Gly Ala Gln Ala Thr Tyr ThrAsn Leu Glu Gly Gly 50 55 60 Thr Tyr Glu Ile Ser Trp Gly Asp Gly Gly AsnLeu Val Gly Gly Lys 65 70 75 80 Gly Trp Asn Pro Gly Leu Asn Ala Arg AlaIle His Phe Glu Gly Val 85 90 95 Tyr Gln Pro Asn Gly Asn Ser Tyr Leu AlaVal Tyr Gly Trp Thr Arg 100 105 110 Asn Pro Leu Val Glu Tyr Tyr Ile ValGlu Asn Phe Gly Thr Tyr Asp 115 120 125 Pro Ser Ser Gly Ala Thr Asp LeuGly Thr Val Glu Cys Asp Gly Ser 130 135 140 Ile Tyr Arg Leu Gly Lys ThrThr Arg Val Asn Ala Pro Ser Ile Asp 145 150 155 160 Gly Thr Gln Thr PheAsp Gln Tyr Trp Ser Val Arg Gln Asp Lys Arg 165 170 175 Thr Ser Gly ThrVal Gln Thr Gly Cys His Phe Asp Ala Trp Ala Arg 180 185 190 Ala Gly LeuAsn Val Asn Gly Asp His Tyr Tyr Gln Ile Val Ala Thr 195 200 205 Glu GlyTyr Phe Ser Ser Gly Tyr Ala Arg Ile Thr Val Ala Asp Val 210 215 220 Gly225 25 231 PRT C. carbonum 25 Met Val Ser Phe Lys Ser Leu Leu Leu AlaAla Val Ala Thr Thr Ser 1 5 10 15 Val Leu Ala Ala Pro Phe Asp Phe LeuArg Glu Arg Asp Asp Val Asn 20 25 30 Ala Thr Ala Leu Leu Glu Lys Arg GlnSer Thr Pro Ser Ala Glu Gly 35 40 45 Tyr His Asn Gly Tyr Phe Tyr Ser TrpTrp Thr Asp Gly Gly Gly Ser 50 55 60 Ala Gln Tyr Thr Met Gly Glu Gly SerArg Tyr Ser Val Thr Trp Arg 65 70 75 80 Asn Thr Gly Asn Phe Val Gly GlyLys Gly Trp Asn Pro Gly Ser Gly 85 90 95 Arg Val Ile Asn Tyr Gly Gly AlaPhe Asn Pro Gln Gly Asn Gly Tyr 100 105 110 Leu Ala Val Tyr Gly Trp ThrArg Asn Pro Leu Val Glu Tyr Tyr Val 115 120 125 Ile Glu Ser Tyr Gly ThrTyr Asn Pro Ser Ser Gly Ala Gln Ile Lys 130 135 140 Gly Ser Phe Gln ThrAsp Gly Gly Thr Tyr Asn Val Ala Val Ser Thr 145 150 155 160 Arg Tyr AsnGln Pro Ser Ile Asp Gly Thr Arg Thr Phe Gln Gln Tyr 165 170 175 Trp SerVal Arg Thr Gln Lys Arg Val Gly Gly Ser Val Asn Met Gln 180 185 190 AsnHis Phe Asn Ala Trp Ser Arg Tyr Gly Leu Asn Leu Gly Gln His 195 200 205Tyr Tyr Gln Ile Val Ala Thr Glu Gly Tyr Gln Ser Ser Gly Ser Ser 210 215220 Asp Ile Tyr Val Gln Thr Gln 225 230 26 231 PRT C. sativus 26 Met ValSer Phe Lys Ser Leu Leu Leu Ala Ala Val Ala Thr Thr Ser 1 5 10 15 ValLeu Ala Ala Pro Phe Asp Phe Leu Arg Glu Arg Asp Asp Gly Asn 20 25 30 AlaThr Ala Leu Leu Glu Lys Arg Gln Ser Thr Pro Ser Ser Glu Gly 35 40 45 TyrHis Asn Gly Tyr Phe Tyr Ser Trp Trp Thr Asp Gly Gly Gly Ser 50 55 60 AlaGln Tyr Thr Met Gly Glu Gly Ser Arg Tyr Ser Val Thr Trp Arg 65 70 75 80Asn Thr Gly Asn Phe Val Gly Gly Lys Gly Trp Asn Pro Gly Thr Gly 85 90 95Arg Val Ile Asn Tyr Gly Gly Ala Phe Asn Pro Gln Gly Asn Gly Tyr 100 105110 Leu Ala Val Tyr Gly Trp Thr Arg Asn Pro Leu Val Glu Tyr Tyr Val 115120 125 Ile Glu Ser Tyr Gly Thr Tyr Asn Pro Ser Ser Gly Ala Gln Val Lys130 135 140 Gly Ser Phe Gln Thr Asp Gly Gly Thr Tyr Asn Val Ala Val SerThr 145 150 155 160 Arg Tyr Asn Gln Pro Ser Ile Asp Gly Thr Arg Thr PheGln Gln Tyr 165 170 175 Trp Ser Val Arg Gln Gln Lys Arg Val Gly Gly SerVal Asn Met Gln 180 185 190 Asn His Phe Asn Ala Trp Ser Arg Tyr Gly LeuAsn Leu Gly Gln His 195 200 205 Tyr Tyr Gln Ile Val Ala Thr Glu Gly TyrGln Ser Ser Gly Ser Ser 210 215 220 Asp Ile Tyr Val Gln Thr Gln 225 23027 227 PRT H. insolens 27 Met Val Ser Leu Lys Ser Val Leu Ala Ala AlaThr Ala Val Ser Ser 1 5 10 15 Ala Ile Ala Ala Pro Phe Asp Phe Val ProArg Asp Asn Ser Thr Ala 20 25 30 Leu Gln Ala Arg Gln Val Thr Pro Asn AlaGlu Gly Trp His Asn Gly 35 40 45 Tyr Phe Tyr Ser Trp Trp Ser Asp Gly GlyGly Gln Val Gln Tyr Thr 50 55 60 Asn Leu Glu Gly Ser Arg Tyr Gln Val ArgTrp Arg Asn Thr Gly Asn 65 70 75 80 Phe Val Gly Gly Lys Gly Trp Asn ProGly Thr Gly Arg Thr Ile Asn 85 90 95 Tyr Gly Gly Tyr Phe Asn Pro Gln GlyAsn Gly Tyr Leu Ala Val Tyr 100 105 110 Gly Trp Thr Arg Asn Pro Leu ValGlu Tyr Tyr Val Ile Glu Ser Tyr 115 120 125 Gly Thr Tyr Asn Pro Gly SerGln Ala Gln Tyr Lys Gly Thr Phe Tyr 130 135 140 Thr Asp Gly Asp Gln TyrAsp Ile Phe Val Ser Thr Arg Tyr Asn Gln 145 150 155 160 Pro Ser Ile AspGly Thr Arg Thr Phe Gln Gln Tyr Trp Ser Ile Arg 165 170 175 Lys Asn LysArg Val Gly Gly Ser Val Asn Met Gln Asn His Phe Asn 180 185 190 Ala TrpGln Gln His Gly Met Pro Leu Gly Gln His Tyr Tyr Gln Val 195 200 205 ValAla Thr Glu Gly Tyr Gln Ser Ser Gly Glu Ser Asp Ile Tyr Val 210 215 220Gln Thr His 225 28 233 PRT M. grisea 28 Met Val Ser Phe Thr Ser Ile ValThr Ala Val Val Ala Leu Ala Gly 1 5 10 15 Ser Ala Leu Ala Ile Pro AlaPro Asp Gly Asn Met Thr Gly Phe Pro 20 25 30 Phe Glu Gln Leu Met Arg ArgGln Ser Thr Pro Ser Ser Thr Gly Arg 35 40 45 His Asn Gly Tyr Tyr Tyr SerTrp Trp Thr Asp Gly Ala Ser Pro Val 50 55 60 Gln Tyr Gln Asn Gly Asn GlyGly Ser Tyr Ser Val Gln Trp Gln Ser 65 70 75 80 Gly Gly Asn Phe Val GlyGly Lys Gly Trp Met Pro Gly Gly Ser Lys 85 90 95 Ser Ile Thr Tyr Ser GlyThr Phe Asn Pro Val Asn Asn Gly Asn Ala 100 105 110 Tyr Leu Cys Ile TyrGly Trp Thr Gln Asn Pro Leu Val Glu Tyr Tyr 115 120 125 Ile Leu Glu AsnTyr Gly Glu Tyr Asn Pro Gly Asn Ser Ala Gln Ser 130 135 140 Arg Gly ThrLeu Gln Ala Ala Gly Gly Thr Tyr Thr Leu His Glu Ser 145 150 155 160 ThrArg Val Asn Gln Pro Ser Ile Glu Gly Thr Arg Thr Phe Gln Gln 165 170 175Tyr Trp Ala Ile Arg Gln Gln Lys Arg Asn Ser Gly Thr Val Asn Thr 180 185190 Gly Glu Phe Phe Gln Ala Trp Glu Arg Ala Gly Met Arg Met Gly Asn 195200 205 His Asn Tyr Met Ile Val Ala Thr Glu Gly Tyr Arg Ser Ala Gly Asn210 215 220 Ser Asn Ile Asn Val Gln Thr Pro Ala 225 230 29 219 PRT C.gracile 29 Met Val Ser Phe Lys Ala Leu Leu Leu Gly Ala Ala Gly Ala LeuAla 1 5 10 15 Phe Pro Phe Asn Val Thr Gln Met Asn Glu Leu Val Ala ArgAla Gly 20 25 30 Thr Pro Ser Gly Thr Gly Thr Asn Asn Gly Tyr Phe Tyr SerPhe Trp 35 40 45 Thr Asp Gly Gly Gly Thr Val Asn Tyr Gln Asn Gly Ala GlyGly Ser 50 55 60 Tyr Ser Val Gln Trp Gln Asn Cys Gly Asn Phe Val Gly GlyLys Gly 65 70 75 80 Trp Asn Pro Gly Ala Ala Arg Thr Ile Asn Phe Ser GlyThr Phe Ser 85 90 95 Pro Gln Gly Asn Gly Tyr Leu Ala Ile Tyr Gly Trp ThrGln Asn Pro 100 105 110 Leu Val Glu Tyr Tyr Ile Val Glu Ser Phe Gly ThrTyr Asp Pro Ser 115 120 125 Ser Gln Ala Ser Lys Phe Gly Thr Ile Gln GlnAsp Gly Ser Thr Tyr 130 135 140 Thr Ile Ala Lys Thr Thr Arg Val Asn GlnPro Ser Ile Glu Gly Thr 145 150 155 160 Ser Thr Phe Asp Gln Phe Trp SerVal Arg Gln Asn His Arg Ser Ser 165 170 175 Gly Ser Val Asn Val Ala AlaHis Phe Asn Ala Trp Ala Gln Ala Gly 180 185 190 Leu Lys Leu Gly Ser HisAsn Tyr Gln Ile Val Ala Thr Glu Gly Tyr 195 200 205 Gln Ser Ser Gly SerSer Ser Ile Thr Val Ser 210 215 30 223 PRT T. reesei 30 Met Val Ser PheThr Ser Leu Leu Ala Gly Val Ala Ala Ile Ser Gly 1 5 10 15 Val Leu AlaAla Pro Ala Ala Glu Val Glu Pro Val Ala Val Glu Lys 20 25 30 Arg Gln ThrIle Gln Pro Gly Thr Gly Tyr Asn Asn Gly Tyr Phe His 35 40 45 Ser Tyr TrpAsn Asp Gly His Gly Gly Val Thr Tyr Thr Asn Gly Pro 50 55 60 Gly Gly GlnPhe Ser Val Asn Trp Ser Asn Ser Gly Asn Phe Val Gly 65 70 75 80 Gly LysGly Trp Gln Pro Gly Thr Lys Asn Lys Val Ile Asn Phe Ser 85 90 95 Gly SerTyr Asn Pro Asn Gly Asn Ser Tyr Leu Ser Val Tyr Gly Trp 100 105 110 SerArg Asn Pro Leu Ile Glu Tyr Tyr Ile Val Gly Asn Phe Gly Thr 115 120 125Tyr Asn Pro Ser Thr Gly Ala Thr Lys Leu Gly Glu Val Thr Ser Asp 130 135140 Gly Ser Val Tyr Asp Ile Tyr Arg Thr Gln Arg Val Asn Gln Pro Ser 145150 155 160 Ile Ile Gly Thr Ala Thr Phe Tyr Gln Tyr Trp Ser Val Arg ArgAsn 165 170 175 His Arg Ser Ser Gly Ser Val Asn Thr Ala Asn His Phe AsnAla Trp 180 185 190 Ala Gln Gln Gly Leu Thr Leu Gly Thr Met Asp Tyr GlnIle Val Ala 195 200 205 Val Glu Gly Tyr Phe Ser Ser Gly Ser Ala Ser IleThr Val Ser 210 215 220 31 223 PRT T. reesei 31 Met Val Ser Phe Thr SerLeu Leu Ala Gly Val Ala Ala Ile Ser Gly 1 5 10 15 Val Leu Ala Ala ProAla Ala Glu Val Glu Ser Val Ala Val Glu Lys 20 25 30 Arg Gln Thr Ile GlnPro Gly Thr Gly Tyr Asn Asn Gly Tyr Phe Tyr 35 40 45 Ser Tyr Trp Asn AspGly His Gly Gly Val Thr Tyr Thr Asn Gly Pro 50 55 60 Gly Gly Gln Phe SerVal Asn Trp Ser Asn Ser Gly Asn Phe Val Gly 65 70 75 80 Gly Lys Gly TrpGln Pro Gly Thr Lys Asn Lys Val Ile Asn Phe Ser 85 90 95 Gly Ser Tyr AsnPro Asn Gly Asn Ser Tyr Leu Ser Val Tyr Gly Trp 100 105 110 Ser Arg AsnPro Leu Ile Glu Tyr Tyr Ile Val Glu Asn Phe Gly Thr 115 120 125 Tyr AsnPro Ser Thr Gly Ala Thr Lys Leu Gly Glu Val Thr Ser Asp 130 135 140 GlySer Val Tyr Asp Ile Tyr Arg Thr Gln Arg Val Asn Gln Pro Ser 145 150 155160 Ile Ile Gly Thr Ala Thr Phe Tyr Gln Tyr Trp Ser Val Arg Arg Asn 165170 175 His Arg Ser Ser Gly Ser Val Asn Thr Ala Asn His Phe Asn Ala Trp180 185 190 Ala Gln Gln Gly Leu Thr Leu Gly Thr Met Asp Tyr Gln Ile ValAla 195 200 205 Val Glu Gly Tyr Phe Ser Ser Gly Ser Ala Ser Ile Thr ValSer 210 215 220 32 222 PRT T. reesei 32 Met Val Ser Phe Thr Ser Leu LeuAla Ala Ser Pro Pro Ser Arg Ala 1 5 10 15 Ser Cys Arg Pro Ala Ala GluVal Glu Ser Val Ala Val Glu Lys Arg 20 25 30 Gln Thr Ile Gln Pro Gly ThrGly Tyr Asn Asn Gly Tyr Phe Tyr Ser 35 40 45 Tyr Trp Asn Asp Gly His GlyGly Val Thr Tyr Thr Asn Gly Pro Gly 50 55 60 Gly Gln Phe Ser Val Asn TrpSer Asn Ser Gly Asn Phe Val Gly Gly 65 70 75 80 Lys Gly Trp Gln Pro GlyThr Lys Asn Lys Val Ile Asn Phe Ser Gly 85 90 95 Ser Tyr Asn Pro Asn GlyAsn Ser Tyr Leu Ser Val Tyr Gly Trp Ser 100 105 110 Arg Asn Pro Leu IleGlu Tyr Tyr Ile Val Glu Asn Phe Gly Thr Tyr 115 120 125 Asn Pro Ser ThrGly Ala Thr Lys Leu Gly Glu Val Thr Ser Asp Gly 130 135 140 Ser Val TyrAsp Ile Tyr Arg Thr Gln Arg Val Asn Gln Pro Ser Ile 145 150 155 160 IleGly Thr Ala Thr Phe Tyr Gln Tyr Trp Ser Val Arg Arg Asn His 165 170 175Arg Ser Ser Gly Ser Val Asn Thr Ala Asn His Phe Asn Ala Trp Ala 180 185190 Gln Gln Gly Leu Thr Leu Gly Thr Met Asp Tyr Gln Ile Val Ala Val 195200 205 Glu Gly Tyr Phe Ser Ser Gly Ser Ala Ser Ile Thr Val Ser 210 215220 33 190 PRT T. harzianum 33 Gln Thr Ile Gly Pro Gly Thr Gly Tyr SerAsn Gly Tyr Tyr Tyr Ser 1 5 10 15 Tyr Trp Asn Asp Gly His Ala Gly ValThr Tyr Thr Asn Gly Gly Gly 20 25 30 Gly Ser Phe Thr Val Asn Trp Ser AsnSer Gly Asn Phe Val Ala Gly 35 40 45 Lys Gly Trp Gln Pro Gly Thr Lys AsnLys Val Ile Asn Phe Ser Gly 50 55 60 Ser Tyr Asn Pro Asn Gly Asn Ser TyrLeu Ser Ile Tyr Gly Trp Ser 65 70 75 80 Arg Asn Pro Leu Ile Glu Tyr TyrIle Val Glu Asn Phe Gly Thr Tyr 85 90 95 Asn Pro Ser Thr Gly Ala Thr LysLeu Gly Glu Val Thr Ser Asp Gly 100 105 110 Ser Val Tyr Asp Ile Tyr ArgThr Gln Arg Val Asn Gln Pro Ser Ile 115 120 125 Ile Gly Thr Ala Thr PheTyr Gln Tyr Trp Ser Val Arg Arg Asn His 130 135 140 Arg Ser Ser Gly SerVal Asn Thr Ala Asn His Phe Asn Ala Trp Ala 145 150 155 160 Ser His GlyLeu Thr Leu Gly Thr Met Asp Tyr Gln Ile Val Ala Val 165 170 175 Glu GlyTyr Phe Ser Ser Gly Ser Ala Ser Ile Thr Val Ser 180 185 190 34 223 PRTT. viride 34 Met Val Ser Phe Thr Thr Leu Leu Ala Gly Phe Val Ala Val ThrGly 1 5 10 15 Val Leu Ser Ala Pro Thr Glu Thr Val Glu Val Val Asp ValGlu Lys 20 25 30 Arg Gln Thr Ile Gly Pro Gly Thr Gly Phe Asn Asn Gly TyrTyr Tyr 35 40 45 Ser Tyr Trp Asn Asp Gly His Ser Gly Val Thr Tyr Thr AsnGly Ala 50 55 60 Gly Gly Ser Phe Ser Val Asn Trp Ala Asn Ser Gly Asn PheVal Gly 65 70 75 80 Gly Lys Gly Trp Asn Pro Gly Ser Ser Ser Arg Val IleAsn Phe Ser 85 90 95 Gly Ser Tyr Asn Pro Asn Gly Asn Ser Tyr Leu Ser ValTyr Gly Trp 100 105 110 Ser Lys Asn Pro Leu Ile Glu Tyr Tyr Ile Val GluAsn Phe Gly Thr 115 120 125 Tyr Asn Pro Ser Thr Gly Thr Thr Lys Leu GlyGlu Val Thr Ser Asp 130 135 140 Gly Ser Val Tyr Asp Ile Tyr Arg Thr GlnArg Val Asn Gln Pro Ser 145 150 155 160 Ile Ile Gly Thr Ala Thr Phe TyrGln Tyr Trp Ser Val Arg Arg Asn 165 170 175 His Ala Pro Ala Ala Arg SerArg Leu Arg Thr Thr Ser Asn Ala Trp 180 185 190 Arg Asn Leu Gly Leu ThrLeu Gly Thr Leu Asp Tyr Gln Ile Ile Ala 195 200 205 Val Glu Gly Tyr PheSer Ser Gly Asn Ala Asn Ile Asn Val Ser 210 215 220 35 241 PRT C.gracile 35 Met Val Asn Phe Ser Ser Leu Phe Leu Ala Ala Ser Ala Ala ValVal 1 5 10 15 Ala Val Ala Ala Pro Gly Glu Leu Pro Gly Met His Lys ArgGln Thr 20 25 30 Leu Thr Ser Ser Gln Thr Gly Thr Asn Asn Gly Tyr Tyr TyrSer Phe 35 40 45 Trp Thr Asp Gly Gln Gly Asn Val Gln Tyr Thr Asn Glu AlaGly Gly 50 55 60 Gln Tyr Ser Val Thr Trp Ser Gly Asn Gly Asn Trp Val GlyGly Lys 65 70 75 80 Gly Trp Asn Pro Gly Ser Ala Arg Thr Ile Asn Tyr ThrAla Asn Tyr 85 90 95 Asn Pro Asn Gly Asn Ser Tyr Leu Ala Val Tyr Gly TrpThr Arg Asn 100 105 110 Pro Leu Ile Glu Tyr Tyr Val Val Glu Asn Phe GlyThr Tyr Asn Pro 115 120 125 Ser Thr Gly Ala Thr Arg Leu Gly Ser Val ThrThr Asp Gly Ser Cys 130 135 140 Tyr Asp Ile Tyr Arg Thr Gln Arg Val AsnGln Pro Ser Ile Glu Gly 145 150 155 160 Thr Ser Thr Phe Tyr Gln Phe TrpSer Val Arg Gln Asn Lys Arg Ser 165 170 175 Gly Gly Ser Val Asn Met AlaAla His Phe Asn Ala Trp Ala Ala Ala 180 185 190 Gly Leu Gln Leu Gly ThrHis Asp Tyr Gln Ile Val Ala Thr Glu Gly 195 200 205 Tyr Tyr Ser Ser GlySer Ala Thr Val Asn Val Gly Ala Ser Ser Asp 210 215 220 Gly Ser Thr GlyGly Gly Ser Thr Gly Gly Gly Ser Thr Asn Val Ser 225 230 235 240 Phe 36225 PRT A. niger 36 Met Leu Thr Lys Asn Leu Leu Leu Cys Phe Ala Ala AlaLys Ala Ala 1 5 10 15 Leu Ala Val Pro His Asp Ser Val Ala Gln Arg SerAsp Ala Leu His 20 25 30 Met Leu Ser Glu Arg Ser Thr Pro Ser Ser Thr GlyGlu Asn Asn Gly 35 40 45 Phe Tyr Tyr Ser Phe Trp Thr Asp Gly Gly Gly AspVal Thr Tyr Thr 50 55 60 Asn Gly Asp Ala Gly Ala Tyr Thr Val Glu Trp SerAsn Val Gly Asn 65 70 75 80 Phe Val Gly Gly Lys Gly Trp Asn Pro Gly SerAla Gln Asp Ile Thr 85 90 95 Tyr Ser Gly Thr Phe Thr Pro Ser Gly Asn GlyTyr Leu Ser Val Tyr 100 105 110 Gly Trp Thr Thr Asp Pro Leu Ile Glu TyrTyr Ile Val Glu Ser Tyr 115 120 125 Gly Asp Tyr Asn Pro Gly Ser Gly GlyThr Tyr Lys Gly Thr Val Thr 130 135 140 Ser Asp Gly Ser Val Tyr Asp IleTyr Thr Ala Thr Arg Thr Asn Ala 145 150 155 160 Ala Ser Ile Gln Gly ThrAla Thr Phe Thr Gln Tyr Trp Ser Val Arg 165 170 175 Gln Asn Lys Arg ValGly Gly Thr Val Thr Thr Ser Asn His Phe Asn 180 185 190 Ala Trp Ala LysLeu Gly Met Asn Leu Gly Thr His Asn Tyr Gln Ile 195 200 205 Val Ala ThrGlu Gly Tyr Gln Ser Ser Gly Ser Ser Ser Ile Thr Val 210 215 220 Gln 22537 221 PRT Penicillium sp 40 37 Met Lys Ser Phe Ile Ala Tyr Leu Leu AlaSer Val Ala Val Thr Gly 1 5 10 15 Val Met Ala Val Pro Gly Glu Tyr HisLys Arg His Asp Lys Arg Gln 20 25 30 Thr Ile Thr Ser Ser Gln Thr Gly ThrAsn Asn Gly Tyr Tyr Tyr Ser 35 40 45 Phe Trp Thr Asn Gly Gly Gly Thr ValGln Tyr Thr Asn Gly Ala Ala 50 55 60 Gly Glu Tyr Ser Val Thr Trp Glu AsnCys Gly Asp Phe Thr Ser Gly 65 70 75 80 Lys Gly Trp Ser Thr Gly Ser AlaArg Asp Ile Thr Phe Glu Gly Thr 85 90 95 Phe Asn Pro Ser Gly Asn Ala TyrLeu Ala Val Tyr Gly Trp Thr Thr 100 105 110 Ser Pro Leu Val Glu Tyr TyrIle Leu Glu Asp Tyr Gly Asp Tyr Asn 115 120 125 Pro Gly Asn Ser Met ThrTyr Lys Gly Thr Val Thr Ser Asp Gly Ser 130 135 140 Val Tyr Asp Ile TyrGlu His Gln Gln Val Asn Gln Pro Ser Ile Ser 145 150 155 160 Gly Thr AlaThr Phe Asn Gln Tyr Trp Ser Ile Arg Gln Asn Thr Arg 165 170 175 Ser SerGly Thr Val Thr Thr Ala Asn His Phe Asn Ala Trp Ala Lys 180 185 190 LeuGly Met Asn Leu Gly Ser Phe Asn Tyr Gln Ile Val Ser Thr Glu 195 200 205Gly Tyr Glu Ser Ser Gly Ser Ser Thr Ile Thr Val Ser 210 215 220 38 240PRT Streptomyces sp 38 Met Gln Gln Asp Gly Lys Arg Gln Asp Gln Asn GlnGln Asn Pro Ala 1 5 10 15 Pro Phe Ser Gly Leu Ser Arg Arg Gly Phe LeuGly Gly Ala Gly Thr 20 25 30 Val Ala Leu Ala Thr Ala Ser Gly Leu Leu LeuPro Ser Thr Ala His 35 40 45 Ala Ala Thr Thr Ile Thr Thr Asn Gln Thr GlyTyr Asp Gly Met Tyr 50 55 60 Tyr Ser Phe Trp Thr Asp Gly Gly Gly Ser ValSer Met Thr Leu Asn 65 70 75 80 Gly Gly Gly Ser Tyr Ser Thr Gln Trp ThrAsn Cys Gly Asn Phe Val 85 90 95 Ala Gly Lys Gly Trp Gly Asn Gly Gly ArgArg Thr Val Arg Tyr Ser 100 105 110 Gly Tyr Phe Asn Pro Ser Gly Asn GlyTyr Gly Cys Leu Tyr Gly Trp 115 120 125 Thr Ser Asn Pro Leu Val Glu TyrTyr Ile Val Asp Asn Trp Gly Ser 130 135 140 Tyr Arg Pro Thr Gly Glu TyrArg Gly Thr Val Tyr Ser Asp Gly Gly 145 150 155 160 Thr Tyr Asp Ile TyrLys Thr Thr Arg Tyr Asn Ala Pro Ser Val Glu 165 170 175 Gly Thr Arg ThrPhe Asp Gln Tyr Trp Ser Val Arg Gln Ser Lys Val 180 185 190 Ile Gly SerGly Thr Ile Thr Thr Gly Asn His Phe Asp Ala Trp Ala 195 200 205 Arg AlaGly Met Asn Leu Gly Gln Phe Gln Tyr Tyr Met Ile Met Ala 210 215 220 ThrGlu Gly Tyr Gln Ser Ser Gly Ser Ser Asn Ile Thr Val Ser Gly 225 230 235240 39 228 PRT Streptomyces sp 39 Met Thr Lys Asp Asn Thr Pro Ile ArgPro Val Ser Arg Arg Gly Phe 1 5 10 15 Ile Gly Arg Ala Gly Ala Leu AlaLeu Ala Thr Ser Gly Leu Met Leu 20 25 30 Pro Gly Thr Ala Arg Ala Asp ThrVal Ile Thr Thr Asn Gln Thr Gly 35 40 45 Thr Asn Asn Gly Tyr Tyr Tyr SerPhe Trp Thr Asp Gly Gly Gly Ser 50 55 60 Val Ser Met Asn Leu Ala Ser GlyGly Ser Tyr Gly Thr Ser Trp Thr 65 70 75 80 Asn Cys Gly Asn Phe Val AlaGly Lys Gly Trp Ala Asn Gly Ala Arg 85 90 95 Arg Thr Val Asn Tyr Ser GlySer Phe Asn Pro Ser Gly Asn Ala Tyr 100 105 110 Leu Thr Leu Tyr Gly TrpThr Ala Asn Pro Leu Val Glu Tyr Tyr Ile 115 120 125 Val Asp Asn Trp GlyThr Tyr Arg Pro Thr Gly Thr Tyr Lys Gly Thr 130 135 140 Val Thr Ser AspGly Gly Thr Tyr Asp Val Tyr Gln Thr Thr Arg Val 145 150 155 160 Asn AlaPro Ser Val Glu Gly Thr Lys Thr Phe Asn Gln Tyr Trp Ser 165 170 175 ValArg Gln Ser Lys Arg Thr Gly Gly Ser Ile Thr Ala Gly Asn His 180 185 190Phe Asp Ala Trp Ala Arg Tyr Gly Met Pro Leu Gly Ser Phe Asn Tyr 195 200205 Tyr Met Ile Met Ala Thr Glu Gly Tyr Gln Ser Ser Gly Ser Ser Ser 210215 220 Ile Ser Val Ser 225 40 239 PRT S. thermocyaneoviolaceus 40 MetAsn Thr Leu Val His Pro Gln Gly Arg Ala Gly Gly Leu Arg Leu 1 5 10 15Leu Val Arg Ala Ala Trp Ala Leu Ala Leu Ala Ala Leu Ala Ala Met 20 25 30Met Phe Gly Gly Thr Ala Arg Ala Asp Thr Ile Thr Ser Asn Gln Thr 35 40 45Gly Thr His Asn Gly Tyr Phe Tyr Ser Phe Trp Thr Asp Ala Pro Gly 50 55 60Thr Val Thr Met Asn Thr Gly Ala Gly Gly Asn Tyr Ser Thr Gln Trp 65 70 7580 Ser Asn Thr Gly Asn Phe Val Ala Gly Lys Gly Trp Ala Thr Gly Gly 85 9095 Arg Arg Thr Val Thr Tyr Ser Gly Thr Phe Asn Pro Ser Gly Asn Ala 100105 110 Tyr Leu Ala Leu Tyr Gly Trp Ser Gln Asn Pro Leu Val Glu Tyr Tyr115 120 125 Ile Val Asp Asn Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr LysGly 130 135 140 Thr Val Tyr Ser Asp Gly Gly Thr Tyr Asp Ile Tyr Met ThrThr Arg 145 150 155 160 Tyr Asn Ala Pro Ser Ile Glu Gly Thr Lys Thr PheAsn Gln Tyr Trp 165 170 175 Ser Val Arg Gln Asn Lys Arg Thr Gly Gly ThrIle Thr Thr Gly Asn 180 185 190 His Phe Asp Ala Trp Ala Ala His Gly MetPro Leu Gly Thr Phe Asn 195 200 205 Tyr Met Ile Leu Ala Thr Glu Gly TyrGln Ser Ser Gly Ser Ser Asn 210 215 220 Ile Thr Val Gly Asp Ser Gly GlyAsp Asn Gly Gly Gly Gly Gly 225 230 235 41 242 PRT S. viridosporus 41Met Asn Ala Phe Ala His Pro Arg Gly Arg Arg His Gly Arg Ser Ala 1 5 1015 Pro Met Ser Pro Arg Ser Thr Trp Ala Val Leu Leu Ala Ala Leu Ala 20 2530 Val Met Leu Leu Pro Gly Thr Ala Thr Ala Ala Pro Val Ile Thr Thr 35 4045 Asn Gln Thr Gly Thr Asn Asn Gly Trp Trp Tyr Ser Phe Trp Thr Asp 50 5560 Ala Gln Gly Thr Val Ser Met Asp Leu Gly Ser Gly Gly Thr Tyr Ser 65 7075 80 Thr Gln Trp Arg Asn Thr Gly Asn Phe Val Ala Gly Lys Gly Trp Ser 8590 95 Thr Gly Gly Arg Lys Thr Val Asn Tyr Ser Gly Thr Phe Asn Pro Ser100 105 110 Gly Asn Ala Tyr Leu Thr Leu Tyr Gly Trp Thr Thr Gly Pro LeuIle 115 120 125 Glu Tyr Tyr Ile Val Asp Asn Trp Gly Thr Tyr Arg Pro ThrGly Lys 130 135 140 Tyr Lys Gly Thr Val Thr Ser Asp Gly Gly Thr Tyr AspIle Tyr Lys 145 150 155 160 Thr Thr Arg Tyr Asn Ala Pro Ser Ile Glu GlyThr Lys Thr Phe Asp 165 170 175 Gln Tyr Trp Ser Val Arg Gln Ser Lys ArgThr Gly Gly Thr Ile Thr 180 185 190 Ser Gly Asn His Phe Asp Ala Trp AlaArg Asn Gly Met Asn Leu Gly 195 200 205 Asn His Asn Tyr Met Ile Met AlaThr Glu Gly Tyr Gln Ser Ser Gly 210 215 220 Ser Ser Thr Ile Thr Val SerGlu Ser Gly Ser Gly Gly Gly Gly Gly 225 230 235 240 Gly Gly 42 240 PRTT. fusca 42 Met Asn His Ala Pro Ala Ser Leu Lys Ser Arg Arg Arg Phe ArgPro 1 5 10 15 Arg Leu Leu Ile Gly Lys Ala Phe Ala Ala Ala Leu Val AlaVal Val 20 25 30 Thr Met Ile Pro Ser Thr Ala Ala His Ala Ala Val Thr SerAsn Glu 35 40 45 Thr Gly Tyr His Asp Gly Tyr Phe Tyr Ser Phe Trp Thr AspAla Pro 50 55 60 Gly Thr Val Ser Met Glu Leu Gly Pro Gly Gly Asn Tyr SerThr Ser 65 70 75 80 Trp Arg Asn Thr Gly Asn Phe Val Ala Gly Lys Gly TrpAla Thr Gly 85 90 95 Gly Arg Arg Thr Val Thr Tyr Ser Ala Ser Phe Asn ProSer Gly Asn 100 105 110 Ala Tyr Leu Thr Leu Tyr Gly Trp Thr Arg Asn ProLeu Val Glu Tyr 115 120 125 Tyr Ile Val Glu Ser Trp Gly Thr Tyr Arg ProThr Gly Thr Tyr Met 130 135 140 Gly Thr Val Thr Thr Asp Gly Gly Thr TyrAsp Ile Tyr Lys Thr Thr 145 150 155 160 Arg Tyr Asn Ala Pro Ser Ile GluGly Thr Arg Thr Phe Asp Gln Tyr 165 170 175 Trp Ser Val Arg Gln Ser LysArg Thr Ser Gly Thr Ile Thr Ala Gly 180 185 190 Asn His Phe Asp Ala TrpAla Arg His Gly Met His Leu Gly Thr His 195 200 205 Asp Tyr Met Ile MetAla Thr Glu Gly Tyr Gln Ser Ser Gly Ser Ser 210 215 220 Asn Val Thr LeuGly Thr Ser Gly Gly Gly Asn Pro Gly Gly Gly Asn 225 230 235 240 43 241PRT C. pachnodae 43 Met Thr Arg Thr Ile Ser Arg Ala Ala His Arg Pro ProAla Gly Gly 1 5 10 15 Arg Ile Ala Arg Ala Leu Ala Ala Ala Gly Ala ThrVal Ala Met Val 20 25 30 Ile Ala Gly Val Ala Ala Ala Gln Pro Ala Ala AlaVal Asp Ser Asn 35 40 45 Ser Thr Gly Ser Ser Gly Gly Tyr Phe Tyr Ser PheTrp Thr Asp Ala 50 55 60 Pro Gly Thr Val Ser Met Asn Leu Gly Ser Gly GlyAsn Tyr Ser Thr 65 70 75 80 Ser Trp Ser Asn Thr Gly Asn Phe Val Ala GlyLys Gly Trp Ser Thr 85 90 95 Gly Ser Ala Arg Thr Ile Ser Tyr Ser Gly ThrPhe Asn Pro Ser Gly 100 105 110 Asn Ala Tyr Leu Ala Val Tyr Gly Trp SerHis Asp Pro Leu Val Glu 115 120 125 Tyr Tyr Ile Val Asp Ser Trp Gly ThrTyr Arg Pro Thr Gly Thr Phe 130 135 140 Met Gly Thr Val Asn Ser Asp GlyGly Thr Tyr Asp Ile Tyr Lys Thr 145 150 155 160 Thr Arg Thr Asn Ala ProSer Ile Glu Gly Thr Ala Thr Phe Thr Gln 165 170 175 Tyr Trp Ser Val ArgGln Ser Lys Arg Val Gly Gly Thr Ile Thr Thr 180 185 190 Ala Asn His PheAsn Ala Trp Ala Ser His Gly Met Asn Leu Gly Arg 195 200 205 His Asp TyrGln Ile Leu Ala Thr Glu Gly Tyr Gln Ser Ser Gly Ser 210 215 220 Ser AsnIle Thr Ile Gly Ser Thr Ser Gly Gly Gly Gly Ser Gly Gly 225 230 235 240Gly 44 221 PRT A. oryzae 44 Met Val Ser Phe Ser Ser Leu Leu Leu Ala ValSer Ala Val Ser Gly 1 5 10 15 Ala Leu Ala Ala Pro Gly Asp Ser Thr LeuVal Glu Leu Ala Lys Arg 20 25 30 Ala Ile Thr Ser Ser Glu Thr Gly Thr AsnAsn Gly Tyr Tyr Tyr Ser 35 40 45 Phe Trp Thr Asn Gly Gly Gly Asp Val GluTyr Thr Asn Gly Asn Gly 50 55 60 Gly Gln Tyr Ser Val Lys Trp Thr Asn CysAsp Asn Phe Val Ala Gly 65 70 75 80 Lys Gly Trp Asn Pro Gly Ser Ala LysThr Val Thr Tyr Ser Gly Glu 85 90 95 Trp Glu Ser Asn Ser Asn Ser Tyr ValSer Leu Tyr Gly Trp Thr Gln 100 105 110 Asn Pro Leu Val Glu Tyr Tyr IleVal Asp Lys Tyr Gly Asp Tyr Asp 115 120 125 Pro Ser Thr Gly Ala Thr GluLeu Gly Thr Val Glu Ser Asp Gly Gly 130 135 140 Thr Tyr Lys Ile Tyr LysThr Thr Arg Glu Asn Ala Pro Ser Ile Glu 145 150 155 160 Gly Thr Ser ThrPhe Asn Gln Tyr Trp Ser Val Arg Gln Ser Gly Arg 165 170 175 Val Gly GlyThr Ile Thr Ala Gln Asn His Phe Asp Ala Trp Ala Asn 180 185 190 Val GlyLeu Gln Leu Gly Thr His Asn Tyr Met Ile Leu Ala Thr Glu 195 200 205 GlyTyr Lys Ser Ser Gly Ser Ala Thr Ile Thr Val Glu 210 215 220 45 216 PRTC. purpurea 45 Met Phe Leu Thr Ser Val Val Ser Leu Val Val Gly Ala IleSer Cys 1 5 10 15 Val Ser Ala Ala Pro Ala Ala Ala Ser Glu Leu Met GlnMet Thr Pro 20 25 30 Arg Asn Ser Cys Tyr Gly Gly Gly Leu Tyr Ser Ser TyrTrp Ala Asp 35 40 45 Tyr Gly Asn Thr Arg Tyr Ser Cys Gly Ala Gly Gly HisTyr Asp Leu 50 55 60 Ser Trp Gly Asn Gly Gly Asn Val Val Ala Gly Arg GlyTrp Lys Pro 65 70 75 80 Ala Ser Pro Arg Ala Val Thr Tyr Ser Gly Ser TrpGln Cys Asn Gly 85 90 95 Asn Cys Tyr Leu Ser Val Tyr Gly Trp Thr Ile AsnPro Leu Val Glu 100 105 110 Tyr Tyr Ile Val Glu Asn Tyr Gly Asn Tyr AsnPro Ser Ala Gly Ala 115 120 125 Gln Arg Arg Gly Gln Val Thr Ala Asp GlySer Ile Tyr Asp Ile Tyr 130 135 140 Ile Ser Thr Gln His Asn Gln Pro SerIle Leu Gly Thr Asn Thr Phe 145 150 155 160 His Gln Tyr Trp Ser Ile ArgArg Asn Lys Arg Val Gly Gly Thr Val 165 170 175 Ser Thr Gly Val His PheAsn Ala Trp Arg Ser Leu Gly Met Pro Leu 180 185 190 Gly Thr Tyr Asp TyrMet Ile Val Ala Thr Glu Gly Phe Arg Ser Ser 195 200 205 Gly Ser Ala SerIle Thr Val Ser 210 215 46 236 PRT C. mixtus 46 Met Lys Phe Pro Leu IleGly Lys Ser Thr Leu Ala Ala Leu Phe Cys 1 5 10 15 Ser Ala Leu Leu GlyVal Asn Asn Thr Gln Ala Gln Thr Leu Thr Asn 20 25 30 Asn Ala Thr Gly ThrHis Asn Gly Phe Tyr Tyr Thr Phe Trp Lys Asp 35 40 45 Ser Gly Asp Ala SerMet Gly Leu Gln Ala Gly Gly Arg Tyr Thr Ser 50 55 60 Gln Trp Ser Asn GlyThr Asn Asn Trp Val Gly Gly Lys Gly Trp Asn 65 70 75 80 Pro Gly Gly ProLys Val Val Thr Tyr Ser Gly Ser Tyr Asn Val Asp 85 90 95 Asn Ser Gln AsnSer Tyr Leu Ala Leu Tyr Gly Trp Thr Arg Ser Pro 100 105 110 Leu Ile GluTyr Tyr Val Ile Glu Ser Tyr Gly Ser Tyr Asn Pro Ala 115 120 125 Ser CysSer Gly Gly Thr Asp Tyr Gly Ser Phe Gln Ser Asp Gly Ala 130 135 140 ThrTyr Asn Val Arg Arg Cys Gln Arg Val Gln Gln Pro Ser Ile Asp 145 150 155160 Gly Thr Gln Thr Phe Tyr Gln Tyr Phe Ser Val Arg Ser Pro Lys Lys 165170 175 Gly Phe Gly Gln Ile Ser Gly Thr Ile Thr Thr Ala Asn His Phe Asn180 185 190 Phe Trp Ala Ser Lys Gly Leu Asn Leu Gly Asn His Asp Tyr MetVal 195 200 205 Leu Ala Thr Glu Gly Tyr Gln Ser Arg Gly Ser Ser Asp IleThr Val 210 215 220 Ser Glu Gly Thr Gly Gly Thr Thr Ser Ser Ser Val 225230 235 47 237 PRT P. fluorescens cellulosa 47 Met Lys Leu Pro Thr LeuGly Lys Cys Val Val Arg Thr Leu Met Gly 1 5 10 15 Ala Val Ala Leu GlyAla Ile Ser Val Asn Ala Gln Thr Leu Ser Ser 20 25 30 Asn Ser Thr Gly ThrAsn Asn Gly Phe Tyr Tyr Thr Phe Trp Lys Asp 35 40 45 Ser Gly Asp Ala SerMet Thr Leu Leu Ser Gly Gly Arg Tyr Gln Ser 50 55 60 Ser Trp Gly Asn SerThr Asn Asn Trp Val Gly Gly Lys Gly Trp Asn 65 70 75 80 Pro Gly Asn AsnSer Arg Val Ile Ser Tyr Ser Gly Ser Tyr Gly Val 85 90 95 Asp Ser Ser GlnAsn Ser Tyr Leu Ala Leu Tyr Gly Trp Thr Arg Ser 100 105 110 Pro Leu IleGlu Tyr Tyr Val Ile Glu Ser Tyr Gly Ser Tyr Asn Pro 115 120 125 Ala SerCys Ser Gly Gly Thr Asp Tyr Gly Ser Phe Gln Ser Asp Gly 130 135 140 AlaThr Tyr Asn Val Arg Arg Cys Gln Arg Val Asn Gln Pro Ser Ile 145 150 155160 Asp Gly Thr Gln Thr Phe Tyr Gln Tyr Phe Ser Val Arg Asn Pro Lys 165170 175 Lys Gly Phe Gly Asn Ile Ser Gly Thr Ile Thr Phe Ala Asn His Val180 185 190 Asn Phe Trp Ala Ser Lys Gly Leu Asn Leu Gly Asn His Asn TyrGln 195 200 205 Val Leu Ala Thr Glu Gly Tyr Gln Ser Arg Gly Ser Ser AspIle Thr 210 215 220 Val Ser Glu Gly Thr Ser Gly Gly Gly Thr Ser Ser Val225 230 235 48 217 PRT P. cochleariae 48 Met Gln Phe Leu Ile Pro Val ValIle Leu Cys Val Ser Leu Val Asp 1 5 10 15 Ser Gln Lys Val Leu Tyr AsnAsn Glu Ile Gly Phe Asn Asn Gly Phe 20 25 30 Tyr Tyr Ala Phe Trp Lys AspSer Gly Ser Ala Thr Phe Thr Leu Glu 35 40 45 Ser Gly Gly Arg Tyr Ala GlyAsn Trp Thr Thr Ser Thr Asn Asn Trp 50 55 60 Val Gly Gly Lys Gly Trp AsnPro Gly Asn Ser Trp Arg Thr Val Asn 65 70 75 80 Tyr Ser Gly Tyr Tyr GlyIle Asn Glu Tyr Ala Asn Ser Tyr Leu Ser 85 90 95 Leu Tyr Gly Trp Thr ThrAsn Pro Leu Ile Glu Tyr Tyr Val Val Glu 100 105 110 Ser Tyr Gly Ser TyrSer Pro Leu Asn Cys Pro Gly Gly Thr Asp Glu 115 120 125 Gly Ser Phe ThrSer Gly Gly Ala Thr Tyr Gln Val Arg Lys Cys Arg 130 135 140 Arg Thr AsnAla Pro Ser Ile Ile Gly Thr Gln Ser Phe Asp Gln Tyr 145 150 155 160 PheSer Val Arg Thr Pro Lys Lys Gly Phe Gly Gln Val Ser Gly Ser 165 170 175Val Asn Phe Ala Asp His Val Gln Tyr Trp Ala Ser Lys Gly Leu Pro 180 185190 Leu Gly Thr His Ala His Gln Ile Phe Ala Thr Glu Gly Tyr Gln Ser 195200 205 Ser Gly Phe Ala Asp Ile Thr Val Ser 210 215 49 182 PRT A.kawachi 49 Ala Gly Ile Asn Tyr Val Gln Asn Tyr Asn Gly Asn Leu Gly AspPhe 1 5 10 15 Thr Tyr Asp Glu Ser Ala Gly Thr Phe Ser Met Tyr Trp GluAsp Gly 20 25 30 Val Ser Ser Asp Phe Val Val Gly Leu Gly Trp Thr Thr GlySer Ser 35 40 45 Asn Ala Ile Thr Tyr Ser Ala Glu Tyr Ser Ala Ser Gly SerSer Ser 50 55 60 Tyr Leu Ala Val Tyr Gly Trp Val Asn Tyr Pro Gln Ala GluTyr Tyr 65 70 75 80 Ile Val Glu Asp Tyr Gly Asp Tyr Asn Pro Cys Ser SerAla Thr Ser 85 90 95 Leu Gly Thr Val Tyr Ser Asp Gly Ser Thr Tyr Gln ValCys Thr Asp 100 105 110 Thr Arg Thr Asn Glu Pro Ser Ile Thr Gly Thr SerThr Phe Thr Gln 115 120 125 Tyr Phe Ser Val Arg Glu Ser Thr Arg Thr SerGly Thr Val Thr Val 130 135 140 Ala Asn His Phe Asn Phe Trp Ala Gln HisGly Phe Gly Asn Ser Asp 145 150 155 160 Phe Asn Tyr Gln Val Met Ala ValGlu Ala Trp Ser Gly Ala Gly Ser 165 170 175 Ala Ser Val Thr Ile Ser 18050 211 PRT A. niger 50 Met Lys Val Thr Ala Ala Phe Ala Gly Leu Leu ValThr Ala Phe Ala 1 5 10 15 Ala Pro Val Pro Glu Pro Val Leu Val Ser ArgSer Ala Gly Ile Asn 20 25 30 Tyr Val Gln Asn Tyr Asn Gly Asn Leu Gly AspPhe Thr Tyr Asp Glu 35 40 45 Ser Ala Gly Thr Phe Ser Met Tyr Trp Glu AspGly Val Ser Ser Asp 50 55 60 Phe Val Val Gly Leu Gly Trp Thr Thr Gly SerSer Lys Ala Ile Thr 65 70 75 80 Tyr Ser Ala Glu Tyr Ser Ala Ser Gly SerSer Ser Tyr Leu Ala Val 85 90 95 Tyr Gly Trp Val Asn Tyr Pro Gln Ala GluTyr Tyr Ile Val Glu Asp 100 105 110 Tyr Gly Asp Tyr Asn Pro Cys Ser SerAla Thr Ser Leu Gly Thr Val 115 120 125 Tyr Ser Asp Gly Ser Thr Tyr GlnVal Cys Thr Asp Thr Arg Thr Asn 130 135 140 Glu Pro Ser Ile Thr Gly ThrSer Thr Phe Thr Gln Tyr Phe Ser Val 145 150 155 160 Arg Glu Ser Thr ArgThr Ser Gly Thr Val Thr Val Ala Asn His Phe 165 170 175 Asn Phe Trp AlaGln His Gly Phe Gly Asn Ser Asp Phe Asn Tyr Gln 180 185 190 Val Met AlaVal Glu Ala Trp Ser Gly Ala Gly Ser Ala Ser Val Thr 195 200 205 Ile SerSer 210 51 210 PRT A .tubigensis 51 Met Lys Val Thr Ala Ala Phe Ala GlyLeu Leu Val Thr Ala Phe Ala 1 5 10 15 Ala Pro Ala Pro Glu Pro Asp LeuVal Ser Arg Ser Ala Gly Ile Asn 20 25 30 Tyr Val Gln Asn Tyr Asn Gly AsnLeu Gly Asp Phe Thr Tyr Asp Glu 35 40 45 Ser Ala Gly Thr Phe Ser Met TyrTrp Glu Asp Gly Val Ser Ser Asp 50 55 60 Phe Val Val Gly Leu Gly Trp ThrThr Gly Ser Ser Thr Ile Thr Tyr 65 70 75 80 Ser Ala Glu Tyr Ser Ala SerGly Ser Ala Ser Tyr Leu Ala Val Tyr 85 90 95 Gly Trp Val Asn Tyr Pro GlnAla Glu Tyr Tyr Ile Val Glu Asp Tyr 100 105 110 Gly Asp Tyr Asn Pro CysSer Ser Ala Thr Ser Leu Gly Thr Val Tyr 115 120 125 Ser Asp Gly Ser ThrTyr Gln Val Cys Thr Asp Thr Arg Thr Asn Glu 130 135 140 Pro Ser Ile ThrGly Thr Ser Thr Phe Thr Gln Tyr Phe Ser Val Arg 145 150 155 160 Glu SerThr Arg Thr Ser Gly Thr Val Thr Val Ala Asn His Phe Asn 165 170 175 PheTrp Ala His His Gly Phe Gly Asn Ser Asp Phe Asn Tyr Gln Val 180 185 190Val Ala Val Glu Ala Trp Ser Gly Ala Gly Ser Ala Ser Val Thr Ile 195 200205 Ser Ser 210 52 208 PRT P. purpurogenum 52 Met Lys Val Thr Ala AlaPhe Ala Gly Leu Leu Ala Arg His Ser Pro 1 5 10 15 Pro Leu Ser Thr GluLeu Val Thr Arg Ser Ile Asn Tyr Val Gln Asn 20 25 30 Tyr Asn Gly Asn LeuGly Ala Phe Ser Tyr Asn Glu Gly Ala Gly Thr 35 40 45 Phe Ser Met Tyr TrpGln Gln Gly Val Ser Asn Asp Phe Val Val Gly 50 55 60 Leu Gly Arg Ser ThrGly Ser Ser Asn Pro Ile Thr Tyr Ser Ala Ser 65 70 75 80 Tyr Ser Ala SerGly Gly Ser Tyr Leu Ala Val Tyr Gly Trp Val Asn 85 90 95 Ser Pro Gln AlaGlu Tyr Tyr Val Val Glu Ala Tyr Gly Asn Tyr Asn 100 105 110 Pro Cys SerSer Gly Ser Ala Thr Asn Leu Gly Thr Val Ser Ser Asp 115 120 125 Gly GlyThr Tyr Gln Val Cys Thr Asp Thr Arg Val Asn Gln Pro Ser 130 135 140 IleThr Gly Thr Ser Thr Phe Thr Gln Phe Phe Ser Val Arg Gln Gly 145 150 155160 Ser Arg Thr Ser Gly Thr Val Thr Ile Ala Asn His Phe Asn Phe Trp 165170 175 Ala Asn Asp Gly Phe Gly Asn Ser Asn Phe Asn Tyr Gln Val Val Ala180 185 190 Val Glu Ala Trp Ser Gly Thr Gly Thr Ala Ser Val Thr Val SerAla 195 200 205 53 233 PRT P. purpurogenum 53 Met Lys Val Thr Ala AlaPhe Ala Gly Leu Leu Ala Arg His Ser Pro 1 5 10 15 Pro Leu Ser Thr GluLeu Val Thr Arg Ser Ile Asn Tyr Val Gln Asn 20 25 30 Tyr Asn Gly Asn LeuGly Ala Phe Ser Tyr Asn Glu Gly Ala Gly Thr 35 40 45 Phe Ser Met Tyr TrpGln Gln Gly Val Ser Asn Asp Phe Val Val Gly 50 55 60 Leu Gly Arg Ser ThrGly Ser Ser Asn Pro Ile Thr Tyr Ser Ala Ser 65 70 75 80 Tyr Ser Ala SerGly Gly Ser Tyr Leu Ala Val Tyr Gly Trp Val Asn 85 90 95 Ser Pro Gln AlaGlu Tyr Tyr Val Val Glu Ala Tyr Gly Asn Tyr Asn 100 105 110 Pro Cys SerSer Gly Ser Ala Thr Asn Leu Gly Thr Val Ser Ser Asp 115 120 125 Gly GlyThr Tyr Gln Val Cys Thr Asp Thr Arg Val Asn Gln Pro Ser 130 135 140 IleThr Gly Thr Ser Thr Phe Thr Gln Phe Phe Ser Val Arg Gln Gly 145 150 155160 Ser Arg Thr Ser Gly Thr Val Thr Ile Ala Asn His Phe Asn Phe Trp 165170 175 Ala Asn Asp Gly Phe Gly Asn Ser Asn Phe Asn Tyr Gln Val Val Ala180 185 190 Val Glu Ala Trp Ser Gly Thr Gly Thr Ala Ser Val Thr Val SerAla 195 200 205 Asn Phe Asn Tyr Gln Val Leu Ala Val Glu Gly Phe Ser GlySer Gly 210 215 220 Asn Ala Asn Met Lys Leu Ile Ser Gly 225 230 54 229PRT T. reesei 54 Met Val Ala Phe Ser Ser Leu Ile Cys Ala Leu Thr Ser IleAla Ser 1 5 10 15 Thr Leu Ala Met Pro Thr Gly Leu Glu Pro Glu Ser SerVal Asn Val 20 25 30 Thr Glu Arg Gly Met Tyr Asp Phe Val Leu Gly Ala HisAsn Asp His 35 40 45 Arg Arg Arg Ala Ser Ile Asn Tyr Asp Gln Asn Tyr GlnThr Gly Gly 50 55 60 Gln Val Ser Tyr Ser Pro Ser Asn Thr Gly Phe Ser ValAsn Trp Asn 65 70 75 80 Thr Gln Asp Asp Phe Val Val Gly Val Gly Trp ThrThr Gly Ser Ser 85 90 95 Ala Pro Ile Asn Phe Gly Gly Ser Phe Ser Val AsnSer Gly Thr Gly 100 105 110 Leu Leu Ser Val Tyr Gly Trp Ser Thr Asn ProLeu Val Glu Tyr Tyr 115 120 125 Ile Met Glu Asp Asn His Asn Tyr Pro AlaGln Gly Thr Val Lys Gly 130 135 140 Thr Val Thr Ser Asp Gly Ala Thr TyrThr Ile Trp Glu Asn Thr Arg 145 150 155 160 Val Asn Glu Pro Ser Ile GlnGly Thr Ala Thr Phe Asn Gln Tyr Ile 165 170 175 Ser Val Arg Asn Ser ProArg Thr Ser Gly Thr Val Thr Val Gln Asn 180 185 190 His Phe Asn Ala TrpAla Ser Leu Gly Leu His Leu Gly Gln Met Asn 195 200 205 Tyr Gln Val ValAla Val Glu Gly Trp Gly Gly Ser Gly Ser Ala Ser 210 215 220 Gln Ser ValSer Asn 225 55 227 PRT B. pumilus 55 Met Asn Leu Lys Arg Leu Arg Leu LeuPhe Val Met Cys Ile Gly Phe 1 5 10 15 Val Leu Thr Leu Thr Ala Val ProAla His Ala Glu Thr Ile Tyr Asp 20 25 30 Asn Arg Ile Gly Thr His Ser GlyTyr Asp Phe Glu Leu Trp Lys Asp 35 40 45 Tyr Gly Asn Thr Ser Met Thr LeuAsn Asn Gly Gly Ala Phe Ser Ala 50 55 60 Ser Trp Asn Asn Ile Gly Asn AlaLeu Phe Arg Lys Gly Lys Lys Phe 65 70 75 80 Asp Ser Thr Lys Thr His HisGln Leu Gly Asn Ile Ser Ile Asn Tyr 85 90 95 Asn Ala Ala Phe Asn Pro GlyGly Asn Ser Tyr Leu Cys Val Tyr Gly 100 105 110 Trp Thr Gln Ser Pro LeuAla Glu Tyr Tyr Ile Val Glu Ser Trp Gly 115 120 125 Thr Tyr Arg Pro ThrGly Thr Tyr Lys Gly Ser Phe Tyr Ala Asp Gly 130 135 140 Gly Thr Tyr AspIle Tyr Glu Thr Leu Arg Val Asn Gln Pro Ser Ile 145 150 155 160 Ile GlyAsp Ala Thr Phe Lys Gln Tyr Trp Ser Val Arg Gln Thr Lys 165 170 175 ArgThr Ser Gly Thr Ala Ser Val Ser Glu His Phe Lys Lys Trp Glu 180 185 190Ser Leu Gly Met Pro Met Gly Lys Met Tyr Glu Thr Ala Leu Thr Val 195 200205 Glu Gly Tyr Arg Ser Asn Gly Ser Ala Asn Val Met Thr Asn Gln Leu 210215 220 Met Ile Arg 225 56 228 PRT B. pumilus 56 Met Asn Leu Arg Lys LeuArg Leu Leu Phe Val Met Cys Ile Gly Leu 1 5 10 15 Thr Leu Ile Leu ThrAla Val Pro Ala His Ala Arg Thr Ile Thr Asn 20 25 30 Asn Glu Met Gly AsnHis Ser Gly Tyr Asp Tyr Glu Leu Trp Lys Asp 35 40 45 Tyr Gly Asn Thr SerMet Thr Leu Asn Asn Gly Gly Ala Phe Ser Ala 50 55 60 Gly Trp Asn Asn IleGly Asn Ala Leu Phe Arg Lys Gly Lys Lys Phe 65 70 75 80 Asp Ser Thr ArgThr His His Gln Leu Gly Asn Ile Ser Ile Asn Tyr 85 90 95 Asn Ala Ser PheAsn Pro Gly Gly Asn Ser Tyr Leu Cys Val Tyr Gly 100 105 110 Trp Thr GlnSer Pro Leu Ala Glu Tyr Tyr Ile Val Asp Ser Trp Gly 115 120 125 Thr TyrArg Pro Thr Gly Ala Tyr Lys Gly Ser Phe Tyr Ala Asp Gly 130 135 140 GlyThr Tyr Asp Ile Tyr Glu Thr Thr Arg Val Asn Gln Pro Ser Ile 145 150 155160 Ile Gly Ile Ala Thr Phe Lys Gln Tyr Trp Ser Val Arg Gln Thr Lys 165170 175 Arg Thr Ser Gly Thr Val Ser Val Ser Ala His Phe Arg Lys Trp Glu180 185 190 Ser Leu Gly Met Pro Met Gly Lys Met Tyr Glu Thr Ala Phe ThrVal 195 200 205 Glu Gly Tyr Gln Ser Ser Gly Ser Ala Asn Val Met Thr AsnGln Leu 210 215 220 Phe Ile Gly Asn 225 57 261 PRT C. acetobutylicum 57Met Leu Arg Arg Lys Val Ile Phe Thr Val Leu Ala Thr Leu Val Met 1 5 1015 Thr Ser Leu Thr Ile Val Asp Asn Thr Ala Phe Ala Ala Thr Asn Leu 20 2530 Asn Thr Thr Glu Ser Thr Phe Ser Lys Glu Val Leu Ser Thr Gln Lys 35 4045 Thr Tyr Ser Ala Phe Asn Thr Gln Ala Ala Pro Lys Thr Ile Thr Ser 50 5560 Asn Glu Ile Gly Val Asn Gly Gly Tyr Asp Tyr Glu Leu Trp Lys Asp 65 7075 80 Tyr Gly Asn Thr Ser Met Thr Leu Lys Asn Gly Gly Ala Phe Ser Cys 8590 95 Gln Trp Ser Asn Ile Gly Asn Ala Leu Phe Arg Lys Gly Lys Lys Phe100 105 110 Asn Asp Thr Gln Thr Tyr Lys Gln Leu Gly Asn Ile Ser Val AsnTyr 115 120 125 Asp Cys Asn Tyr Gln Pro Tyr Gly Asn Ser Tyr Leu Cys ValTyr Gly 130 135 140 Trp Thr Ser Ser Pro Leu Val Glu Tyr Tyr Ile Val AspSer Trp Gly 145 150 155 160 Ser Trp Arg Pro Pro Gly Gly Thr Ser Lys GlyThr Ile Thr Val Asp 165 170 175 Gly Gly Ile Tyr Asp Ile Tyr Glu Thr ThrArg Ile Asn Gln Pro Ser 180 185 190 Ile Gln Gly Asn Thr Thr Phe Lys GlnTyr Trp Ser Val Arg Arg Thr 195 200 205 Lys Arg Thr Ser Gly Thr Ile SerVal Ser Lys His Phe Ala Ala Trp 210 215 220 Glu Ser Lys Gly Met Pro LeuGly Lys Met His Glu Thr Ala Phe Asn 225 230 235 240 Ile Glu Gly Tyr GlnSer Ser Gly Lys Ala Asp Val Asn Ser Met Ser 245 250 255 Ile Asn Ile GlyLys 260 58 234 PRT C. thermocellum 58 Met Lys Gln Lys Leu Leu Val ThrPhe Leu Ile Leu Ile Thr Phe Thr 1 5 10 15 Val Ser Leu Thr Leu Phe ProVal Asn Val Arg Ala Asp Val Val Ile 20 25 30 Thr Ser Asn Gln Thr Gly ThrGly Gly Gly Tyr Asn Phe Glu Tyr Trp 35 40 45 Lys Asp Thr Gly Asn Gly ThrMet Val Leu Lys Asp Gly Gly Ala Phe 50 55 60 Ser Cys Glu Trp Ser Asn IleAsn Asn Ile Leu Phe Arg Lys Gly Phe 65 70 75 80 Lys Tyr Asp Glu Thr LysThr His Asp Gln Leu Gly Tyr Ile Thr Val 85 90 95 Thr Tyr Ser Cys Asn TyrGln Pro Asn Gly Asn Ser Tyr Leu Gly Val 100 105 110 Tyr Gly Trp Thr SerAsn Pro Leu Val Glu Tyr Tyr Ile Ile Glu Ser 115 120 125 Trp Gly Thr TrpArg Pro Pro Gly Ala Thr Pro Lys Gly Thr Ile Thr 130 135 140 Val Asp GlyGly Thr Tyr Glu Ile Tyr Glu Thr Thr Arg Val Asn Gln 145 150 155 160 ProSer Ile Lys Gly Thr Ala Thr Phe Gln Gln Tyr Trp Ser Val Arg 165 170 175Thr Ser Lys Arg Thr Ser Gly Thr Ile Ser Val Thr Glu His Phe Lys 180 185190 Ala Trp Glu Arg Leu Gly Met Lys Met Gly Lys Met Tyr Glu Val Ala 195200 205 Leu Val Val Glu Gly Tyr Gln Ser Ser Gly Lys Ala Asp Val Thr Ser210 215 220 Met Thr Ile Thr Val Gly Asn Ala Pro Ser 225 230 59 230 PRTBacillus sp 41M-1 59 Met Lys Gln Val Lys Ile Met Phe Leu Met Thr Met PheLeu Gly Ile 1 5 10 15 Gly Leu Leu Phe Phe Ser Glu Asn Ala Glu Ala AlaIle Thr Ser Asn 20 25 30 Glu Ile Gly Thr His Asp Gly Tyr Asp Tyr Glu PheTrp Lys Asp Ser 35 40 45 Gly Gly Ser Gly Ser Met Thr Leu Asn Ser Gly GlyThr Phe Ser Ala 50 55 60 Gln Trp Ser Asn Val Asn Asn Ile Leu Phe Arg LysGly Lys Lys Phe 65 70 75 80 Asp Glu Thr Gln Thr His Gln Gln Ile Gly AsnMet Ser Ile Asn Tyr 85 90 95 Gly Ala Thr Tyr Asn Pro Asn Gly Asn Ser TyrLeu Thr Val Tyr Gly 100 105 110 Trp Thr Val Asp Pro Leu Val Glu Phe TyrIle Val Asp Ser Trp Gly 115 120 125 Thr Trp Arg Pro Pro Gly Gly Thr ProLys Gly Thr Ile Asn Val Asp 130 135 140 Gly Gly Thr Tyr Gln Ile Tyr GluThr Thr Arg Tyr Asn Gln Pro Ser 145 150 155 160 Ile Lys Gly Thr Ala ThrPhe Gln Gln Tyr Trp Ser Val Arg Thr Ser 165 170 175 Lys Arg Thr Ser GlyThr Ile Ser Val Ser Glu His Phe Arg Ala Trp 180 185 190 Glu Ser Leu GlyMet Asn Met Gly Asn Met Tyr Glu Val Ala Leu Thr 195 200 205 Val Glu GlyTyr Gln Ser Ser Gly Ser Ala Asn Val Tyr Ser Asn Thr 210 215 220 Leu ThrIle Gly Gly Gln 225 230 60 217 PRT P. multivesiculatum 60 Glu Lys ValIle Cys Leu Leu Ile Ala Leu Phe Gly Leu Ile Glu Ala 1 5 10 15 Gln ThrPhe Tyr Asn Asn Ala Gln Gly Gln Ile Asp Gly Leu Asp Tyr 20 25 30 Glu LeuTrp Lys Asp Thr Gly Thr Thr Ser Met Thr Leu Leu Gly Gly 35 40 45 Gly LysPhe Ser Cys Ser Trp Ser Asn Ile Asn Asn Cys Leu Phe Arg 50 55 60 Ile GlyLys Lys Trp Asn Cys Gln Tyr Glu Trp Trp Glu Leu Gly Thr 65 70 75 80 ValLeu Val Asn Tyr Asp Val Asp Tyr Asn Pro Asn Gly Asn Ser Tyr 85 90 95 LeuCys Ile Tyr Gly Trp Thr Arg Asn Pro Leu Val Glu Tyr Tyr Ile 100 105 110Val Glu Ser Trp Gly Ser Trp Arg Pro Pro Gly Gly Ser Pro Met Asn 115 120125 Thr Met Tyr Val Asp Asp Gly Gln Tyr Asp Val Tyr Val Thr Asp Arg 130135 140 Ile Asn Gln Pro Ser Ile Asp Gly Asn Thr Asn Phe Lys Gln Tyr Trp145 150 155 160 Ser Val Arg Thr Gln Lys Lys Thr Arg Gly Thr Val His ValAsn His 165 170 175 His Phe Tyr Asn Trp Gln Glu Met Gly Leu Lys Val GlyLys Val Tyr 180 185 190 Glu Ala Ser Leu Asn Ile Glu Gly Tyr Gln Ser AlaGly Ser Ala Thr 195 200 205 Val Asn Lys Asn Glu Val Val Gln Thr 210 21561 175 PRT P multivesiculatum 61 Met Thr Leu Leu Gly Gly Gly Lys Phe SerCys Asn Trp Ser Asn Ile 1 5 10 15 Gly Asn Ala Leu Phe Arg Ile Gly LysLys Trp Asp Cys Thr Lys Thr 20 25 30 Trp Gln Gln Leu Gly Thr Ile Ser ValAla Tyr Asn Val Asp Tyr Arg 35 40 45 Pro Asn Gly Asn Ser Tyr Met Cys ValTyr Gly Trp Thr Arg Ser Pro 50 55 60 Leu Ile Glu Tyr Tyr Ile Val Asp SerTrp Gly Ser Trp Arg Pro Pro 65 70 75 80 Gly Ser Asn Ser Met Gly Thr IleAsn Val Asp Gly Gly Thr Tyr Asp 85 90 95 Ile Tyr Val Thr Asp Arg Ile AsnGln Pro Ser Ile Asp Gly Thr Thr 100 105 110 Thr Phe Lys Gln Phe Trp SerVal Arg Thr Gln Lys Lys Thr Ser Gly 115 120 125 Val Ile Ser Val Ser LysHis Phe Glu Ala Trp Thr Ser Lys Gly Leu 130 135 140 Asn Leu Gly Leu MetTyr Glu Ala Ser Leu Thr Ile Glu Gly Tyr Gln 145 150 155 160 Ser Ser GlySer Ala Thr Val Asn Gln Asn Asp Val Thr Gly Gly 165 170 175 62 265 PRTR. albus 62 Met Arg Asn Asn Phe Lys Met Arg Val Met Ala Gly Val Ala AlaVal 1 5 10 15 Ile Cys Leu Ala Gly Val Leu Gly Ser Cys Gly Asn Ser SerAsp Lys 20 25 30 Asp Ser Ser Ser Lys Lys Ser Ala Asp Ser Ala Lys Ala AspSer Asn 35 40 45 Lys Asp Ser Lys Asn Gly Gln Val Phe Thr Lys Asn Ala ArgGly Thr 50 55 60 Ser Asp Gly Tyr Asp Tyr Glu Leu Trp Lys Asp Lys Gly AspThr Glu 65 70 75 80 Met Thr Ile Asn Glu Gly Gly Thr Phe Ser Cys Lys TrpSer Asn Ile 85 90 95 Asn Asn Ala Leu Phe Arg Arg Gly Lys Lys Phe Asp CysThr Lys Thr 100 105 110 Tyr Lys Glu Leu Gly Asn Ile Ser Val Lys Tyr GlyVal Asp Tyr Gln 115 120 125 Pro Asp Gly Asn Ser Tyr Met Cys Val Tyr GlyTrp Thr Ile Asp Pro 130 135 140 Leu Val Glu Phe Tyr Ile Val Glu Ser TrpGly Ser Trp Arg Pro Pro 145 150 155 160 Gly Ala Ala Glu Ser Leu Gly ThrVal Thr Val Asp Gly Gly Thr Tyr 165 170 175 Asp Ile Tyr Lys Thr Thr ArgTyr Glu Gln Pro Ser Ile Asp Gly Thr 180 185 190 Lys Thr Phe Asp Gln TyrTrp Ser Val Arg Gln Asp Lys Pro Thr Gly 195 200 205 Asp Gly Thr Lys IleGlu Gly Thr Ile Ser Ile Ser Lys His Phe Asp 210 215 220 Ala Trp Glu GlnVal Gly Leu Thr Leu Gly Asn Met Tyr Glu Val Ala 225 230 235 240 Leu AsnIle Glu Gly Tyr Gln Ser Asn Gly Gln Ala Thr Ile Tyr Glu 245 250 255 AsnGlu Leu Thr Val Asp Gly Asn Tyr 260 265 63 226 PRT Caldicellulosiruptorsp 63 Met Cys Val Val Leu Ala Asn Pro Phe Tyr Ala Gln Ala Ala Met Thr 15 10 15 Phe Thr Ser Asn Ala Thr Gly Thr Tyr Asp Gly Tyr Tyr Tyr Glu Leu20 25 30 Trp Lys Asp Thr Gly Asn Thr Thr Met Thr Val Asp Thr Gly Gly Arg35 40 45 Phe Ser Cys Gln Trp Ser Asn Ile Asn Asn Ala Leu Phe Arg Thr Gly50 55 60 Lys Lys Phe Ser Thr Ala Trp Asn Gln Leu Gly Thr Val Lys Ile Thr65 70 75 80 Tyr Ser Ala Thr Tyr Asn Pro Asn Gly Asn Ser Tyr Leu Cys IleTyr 85 90 95 Gly Trp Ser Arg Asn Pro Leu Val Glu Phe Tyr Ile Val Glu SerTrp 100 105 110 Gly Ser Trp Arg Pro Pro Gly Ala Thr Ser Leu Gly Thr ValThr Ile 115 120 125 Asp Gly Ala Thr Tyr Asp Ile Tyr Lys Thr Thr Arg ValAsn Gln Pro 130 135 140 Ser Ile Glu Gly Thr Arg Thr Phe Asp Gln Tyr TrpSer Val Arg Thr 145 150 155 160 Ser Lys Arg Thr Ser Gly Thr Val Thr ValThr Asp His Phe Lys Ala 165 170 175 Trp Ala Ala Lys Gly Leu Asn Leu GlyThr Ile Asp Gln Ile Thr Leu 180 185 190 Cys Val Glu Gly Tyr Gln Ser SerGly Ser Ala Asn Ile Thr Gln Asn 195 200 205 Thr Phe Thr Ile Gly Gly SerSer Ser Gly Ser Ser Asn Gly Ser Asn 210 215 220 Asn Gly 225 64 232 PRTD. thermophilum 64 Met Phe Leu Lys Lys Leu Ser Lys Leu Leu Leu Val ValLeu Leu Val 1 5 10 15 Ala Val Tyr Thr Gln Val Asn Ala Gln Thr Ser IleThr Leu Thr Ser 20 25 30 Asn Ala Ser Gly Thr Phe Asp Gly Tyr Tyr Tyr GluLeu Trp Lys Asp 35 40 45 Thr Gly Asn Thr Thr Met Thr Val Tyr Thr Gln GlyArg Phe Ser Cys 50 55 60 Gln Trp Ser Asn Ile Asn Asn Ala Leu Phe Arg ThrGly Lys Lys Tyr 65 70 75 80 Asn Gln Asn Trp Gln Ser Leu Gly Thr Ile ArgIle Thr Tyr Ser Ala 85 90 95 Thr Tyr Asn Pro Asn Gly Asn Ser Tyr Leu CysIle Tyr Gly Trp Ser 100 105 110 Thr Asn Pro Leu Val Glu Phe Tyr Ile ValGlu Ser Trp Gly Asn Trp 115 120 125 Arg Pro Pro Gly Ala Thr Ser Leu GlyGln Val Thr Ile Asp Gly Gly 130 135 140 Thr Tyr Asp Ile Tyr Arg Thr ThrArg Val Asn Gln Pro Ser Ile Val 145 150 155 160 Gly Thr Ala Thr Phe AspGln Tyr Trp Ser Val Arg Thr Ser Lys Arg 165 170 175 Thr Ser Gly Thr ValThr Val Thr Asp His Phe Arg Ala Trp Ala Asn 180 185 190 Arg Gly Leu AsnLeu Gly Thr Ile Asp Gln Ile Thr Leu Cys Val Glu 195 200 205 Gly Tyr GlnSer Ser Gly Ser Ala Asn Ile Thr Gln Asn Thr Phe Ser 210 215 220 Gln GlySer Ser Ser Gly Ser Ser 225 230 65 255 PRT R. flavefaciens 65 Met LysLeu Ser Lys Ile Lys Lys Val Leu Ser Gly Thr Val Ser Ala 1 5 10 15 LeuMet Ile Ala Ser Ala Ala Pro Val Val Ala Ser Ala Ala Asp Gln 20 25 30 GlnThr Arg Gly Asn Val Gly Gly Tyr Asp Tyr Glu Met Trp Asn Gln 35 40 45 AsnGly Gln Gly Gln Ala Ser Met Asn Pro Gly Ala Gly Ser Phe Thr 50 55 60 CysSer Trp Ser Asn Ile Glu Asn Phe Leu Ala Arg Met Gly Lys Asn 65 70 75 80Tyr Asp Ser Gln Lys Lys Asn Tyr Lys Ala Phe Gly Asn Ile Val Leu 85 90 95Thr Tyr Asp Val Glu Tyr Thr Pro Arg Gly Asn Ser Tyr Met Cys Val 100 105110 Tyr Gly Trp Thr Arg Asn Pro Leu Met Glu Tyr Tyr Ile Val Glu Gly 115120 125 Trp Gly Asp Trp Arg Pro Pro Gly Asn Asp Gly Glu Val Lys Gly Thr130 135 140 Val Ser Ala Asn Gly Asn Thr Tyr Asp Ile Arg Lys Thr Met ArgTyr 145 150 155 160 Asn Gln Pro Ser Leu Asp Gly Thr Ala Thr Phe Pro GlnTyr Trp Ser 165 170 175 Val Arg Gln Thr Ser Gly Ser Ala Asn Asn Gln ThrAsn Tyr Met Lys 180 185 190 Gly Thr Ile Asp Val Thr Lys His Phe Asp AlaTrp Ser Ala Ala Gly 195 200 205 Leu Asp Met Ser Gly Thr Leu Tyr Glu ValSer Leu Asn Ile Glu Gly 210 215 220 Tyr Arg Ser Asn Gly Ser Ala Asn ValLys Ser Val Ser Val Thr Gln 225 230 235 240 Gly Gly Ser Ser Asp Asn GlyGly Gln Gln Gln Asn Asn Asp Trp 245 250 255 66 280 PRT P. stipitis 66Met Thr Val Tyr Lys Arg Lys Ser Arg Val Leu Ile Ala Val Val Thr 1 5 1015 Leu Leu His Val Leu Ser His Ala Pro Thr Lys Met Leu Thr Thr Asp 20 2530 Val Leu Leu Thr Arg Cys Met His Leu Cys His Phe Arg Thr Ser Asp 35 4045 Ser Val Tyr Thr Asn Glu Thr Ser Glu Glu Arg Ser Met Ser Asp Arg 50 5560 Leu Asn Ile Thr Arg Val Met Ser Tyr Asp Arg Trp Thr Asp Leu Val 65 7075 80 Gly Glu Leu Glu Val Arg Glu Leu Lys His Val Met Ser His Arg Thr 8590 95 Tyr Ser Leu Cys Asp Leu Ser Cys Ser Thr Val Leu Asp Ser Asn Ser100 105 110 Met Phe Ser Leu Gly Lys Gly Trp Gln Ala Ile Ser Ser Arg GlnGly 115 120 125 Val Gly Ala Thr Val Tyr Gly Trp Thr Arg Ser Pro Leu LeuIle Glu 130 135 140 Tyr Tyr Val Val Asp Ser Trp Gly Ser Tyr His Pro SerAsn Thr Ile 145 150 155 160 Thr Gly Thr Phe Val Thr Val Lys Cys Asp GlyGly Thr Tyr Asp Ile 165 170 175 Tyr Thr Ala Val Arg Val Asn Ala Pro SerIle Glu Gly Thr Thr Phe 180 185 190 Thr Gln Tyr Trp Ser Val Arg Gln SerAla Thr Ile Gln Leu Ala Val 195 200 205 Ile Lys Pro Leu Thr Leu Gln AsnAla Thr Ile Thr Phe Thr Phe Ser 210 215 220 Asn His Phe Asp Ala Trp LysThr Met Thr Leu Glu Ala Thr His Ser 225 230 235 240 Thr Glu Gly Tyr PheSer Ser Gly Ile Thr Tyr Glu Gln Pro His Gln 245 250 255 Pro His Arg AsnThr Trp Ala Thr Ser Leu Thr Ser Gln Thr Lys His 260 265 270 Thr Ala ArgSer Leu Pro Ile Asn 275 280

1. A variant xylanase polypeptide, or fragment thereof having xylanaseactivity, comprising one or more amino acid modifications such that thepolypeptide or fragment thereof has an altered sensitivity to a xylanaseinhibitor as compared with the parent xylanase enzyme.
 2. A variantpolypeptide according to claim 1 which is derived from a family 11xylanase.
 3. A variant xylanase polypeptide, or fragment thereof havingxylanase activity, according to claim 1 or claim 2 wherein said aminoacid modification is of one or more surface amino acid residues.
 4. Avariant xylanase polypeptide, or fragment thereof having xylanaseactivity, according to any one of the preceding claims wherein saidamino acid modification is of one or more solvent accessible residues.5. A variant xylanase polypeptide, or fragment thereof having xylanaseactivity, according to any one of the preceding claims wherein there areat least two of said amino acid modifications.
 6. A variant xylanasepolypeptide, or fragment thereof having xylanase activity according toany one of the preceding claims wherein said amino acid modification isat any one or more of amino acid residues: Ala1-Trp6, Asn8, Thr10-Gly23,Asn25, Ser27, Asn29, Ser31-Asn32, Gly34, Thr43-Thr44, Ser46-Thr50,Asn52, Asn54, Gly56-Asn61, Asn63, Arg73-Leu76, Thr87-Arg89, Thr91-Lys95,Thr97, Lys99, Asp101-Gly102, Thr104, Thr109-Thr111, Tyr113-Asn114,Asp119-Thr124, Thr126, Gln133-Asn141, Thr143, Thr145, Thr147-Asn148,Asn151, Lys154-Gly157, Asn159-Leu160, Ser162-Trp164, Gln175, Ser177,Ser179, Asn181, Thr183, Trp185 of the B. subtilis amino acid sequenceshown as SEQ I.D. No. 1 or its/their equivalent positions in otherhomologous xylanase polypeptides.
 7. A variant xylanase polypeptide, orfragment thereof having xylanase activity, according to any one of thepreceding claims wherein said amino acid modification is at any one ormore of amino acid residues numbers: 11, 12, 13, 15, 17, 29, 31, 32, 34,113, 114, 119, 120, 121, 122, 123, 124 and 175 of the B. subtilis aminoacid sequence shown as SEQ I.D. No. 1 or their equivalent positions inother homologous xylanase polypeptides.
 8. A variant xylanasepolypeptide, or fragment thereof having xylanase activity, according toclaim 7 wherein said variant xylanase polypeptide, or fragment thereofhaving xylanase activity, in addition comprises one or more amino acidmodifications at any one of the other amino acid residues.
 9. A variantxylanase polypeptide, or fragment thereof having xylanase activity,according to claim 8 wherein said other amino acid residues are any oneor more of amino acid residues numbers: 3, 4, 5, 6, 7, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 38, 39, 40, 41, 42, 43, 44, 45, 55, 56, 57, 58,59, 60, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 108, 109,110, 126, 127, 128, 129, 130, 131, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 179, 180, 181, 182, 183 of theB. subtilis amino acid sequence shown as SEQ I.D. No. 1 or theirequivalent positions in other homologous xylanase polypeptides.
 10. Avariant xylanase polypeptide, or fragment thereof having xylanaseactivity, according to claim 8 wherein said other surface amino acidresidues are any one or more of amino acid residues numbers: 1, 2, 46,47, 48, 49, 50, 51, 52, 53, 54, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 184, 185 of the B. subtilis amino acid sequence shown as SEQI.D. No. 1 or their equivalent positions in other homologous xylanasepolypeptides.
 11. A variant polypeptide according to any one of thepreceding claims wherein the inhibitor is an inhibitor found naturallyin plant tissues.
 12. A variant polypeptide according to any one of thepreceding claims wherein the sensitivity to the inhibitor is reduced.13. A method of altering the sensitivity of a xylanase polypeptide to aninhibitor which method comprises modifying one or more amino acidresidues of said enzyme such that the polypeptide or a fragment thereofhas an altered sensitivity to a xylanase inhibitor as compared with theparent xylanase enzyme.
 14. A method according to claim 13 wherein saidvariant polypeptide is that defined in any one of claims 1 to
 12. 15. Amethod according to claim 13 or claim 14 wherein the sensitivity isreduced.
 16. A composition comprising a variant polypeptide according toany one of claims 1 to
 12. 17. A method of degrading or modifying aplant cell wall which method comprises contacting said plant cell wallwith a polypeptide according to any one of claims 1 to 12 or acomposition according to claim
 16. 18. A method of processing a plantmaterial which method comprises contacting said plant material with apolypeptide according to any one of claims 1 to 12 or compositionaccording to claim
 16. 19. A nucleotide sequence encoding a variantpolypeptide according to any one of claims 1 to
 12. 20. A constructcomprising the nucleotide sequence according to claim
 19. 21. Use of avariant polypeptide according to any one of claims 1 to 12 in a methodof modifying plant materials.
 22. Use of a variant polypeptide accordingto any one of claims 1 to 12 in any one or more of: baking, processingcereals, starch production, in processing wood, enhancing the bleachingof wood pulp.